11th ISOBC Abstracts (including Author index)

J. Iran. Chem. Soc., Vol. 9, Suppl. 1, June 2012, pp. A1-A144. JOURNAL OF THE

Iranian Chemical Society

Proceeding Abstracts The First International & 11th Iran Biophysical Chemistry Conference 13-15 June 2012 Ardabil University of Medical Sciences, Ardabil, Iran

Abstract No.1 Keywords: Glucose Oxidase, Circular Dichroism, Nano-Composite, Conformational Change of Glucose Oxidase on Ionic

Ionic Liquid.

Liquid/MWCNTs Nano-composite Abstract No.2

Somayeh Karimi* 1, Hedayatollah Ghourchian 1, Agdas Banaei 2 Triggered Gene Expression Inside Fed-Vesicle Microreactors 1. Institute of Biochemistry and Biophysics, IR

with a Multifunctional Membrane

2. Research Institute of Applied Sciences, IR (E-mail: [email protected])

Zohreh Nourian*, C.J.A. Danelon

Protein conformational changes may be associated with particular

Bionanoscience Department, Kavli Institute, The Netehrlands, NL

properties such as function, transportation, assembly, tendency to

(E-mail: [email protected])

aggregate and potential cytotoxicity. Protein misfolding, in particular, has been intimately related to protein-mediated diseases. In this study,

We established a method to successfully produce lipid vesicles

the conformational structure changes of glucose oxidase (Gox) induced

encapsulating a DNA template and a minimal gene expression system

by the assembly on Ionic liquid (IL)/Carbon nanotube (CNTs) surface

for mRNA and protein synthesis. Our method is compatible with a

were studied in detail by various spectroscopic techniques including

variety of natural and functionalized lipids, which allowed us to

UV-vis absorption, fluorescence and circular dichroism (CD). The

engineer the vesicle membrane for surface immobilization and for

assembly of Gox on the boundary surface of CNTs could result a

promoting selective exchange with the environment. We demonstrated

disturbance of the structure of Gox and induce the exposure of the

that gene expression can be triggered by supplying the nutrients –

prosthetic group and tryptophan (Trp) residues to the solvent, leading

amino acids, nucleotides, tRNAs – in the outside solution. Protein

to a less shielded GOD active site. The result of CD spectra of the

synthesis can take place in liposomes with a broad range of sizes and

Gox/CNTs bioconjugate system showed that CNTs could induce the

the level of expression varies markedly between individual vesicles. We

conversion of α-helix to β-sheet structures and unfolding of the

believe that our results are of direct relevance for initiating, regulating

protein. The results of UV–vis, CD spectra and fluorescence

and monitoring biochemical reaction networks in general, with far-

demonstrate that Gox conjugate with IL/CNTs nano-composites does

reaching consequences to the construction of artificial cells and next

not undergo the structural change and remains its native integrity.

generation drug delivery systems.

Thus ILs is able to generate and maintain an excellent nonconventional environment for proteins. The stability, activity, selectivity

Keywords: Gene Expression, Fed-Vesicle Microreactor, Liposome,

and enantioselectivity of the enzyme which are highly affected by the

Artificial Cell.

nature of IL can be fine tuned by choosing the appropriate cation and anion.

The First International & 11th Iran Biophysical Chemistry Conference, 13-15 June 2012, Ardabil University of Medical Sciences, Ardabil, Iran

Conformational Stability.

Abstract No.3 Comparative Studies on PSH, Drug Binding Characteristics and Conformational Stability of Native and Modified Forms of

Abstract No.4

Bovine Serum Albumin Comparative Studies on Drug Binding Characteristics and

Reza Khodarahmi 1, S.Arash Karimi 2, Mohammad Reza Ashrafi* 3,

Protein Surface Hydrophobicity of Native and Modified forms

Seyyed Abolghasem Ghadami 3, Sirous Ghobadi 4, Mojtaba Amani 5

of Bovine Serum Albumin: Possible Relevance to Change in Protein Structure Upon Glycation

1. Medical Biology Research Center, Departments of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical

Reza Khodarahmi* 1, S.Arash Karimi 2, Mohammad Reza Ashrafi 3,

Sciences, Kermanshah, IR

Seyyed Abolghasem Ghadami 3, Sirous Ghobadi 4, Mojtaba Amani 5

2. Departments of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, IR

1. Medical Biology Research Center, Departments of Pharmacognosy and

3. Medical Biology Research Center, Kermanshah University of Medical

Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical



4. Departments of Biology, Faculty of Sciences, Razi University,

2. Departments of Pharmacognosy and Biotechnology, Faculty of

Kermanshah, IR

Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, IR

5. Faculty of Medicine, Ardabil University of Medical Sciences, Ardabil, IR



Sciences, Kermanshah, IR 4. Departments of Biology, Faculty of Sciences, Razi University,

Interaction of drugs with proteins, especially Serum albumin (SA), has

Kermanshah, IR

a great significance in pharmacology. SA can affect the biological

5. Faculty of Medicine, Ardabil University of Medical Sciences, Ardabil, IR

activity and toxicity of drug. The binding parameters are helpful in the


study of pharmacokinetics and the design of therapeutic dosages. Determination of the impact of various factors such as chemical

The interaction between serum albumin (SA) and drugs has provided

modifications which may occur in vivo is also important when drug

an interesting ground for understanding of drug effects, especially in

binds with albumin to a significant degree. In this study, the effect of

drug distribution and drug–drug interaction on SA, in the case of multi-

some modifications on protein stability, surface hydrophobicity and

drug therapy. Determination of the impact of various factors on drug–

interaction with furosemi dewas investigated by intrinsic and extrinsic

protein interaction is especially important upon significant binding of

fluorescence techniques at physiological conditions. Bovine serum

drug to albumin. In the present study, the interaction of two drugs

albumin (BSA) has been chemically modified with Citraconic anhydride

(furosemide and indomethacin) with native and modified albumins

and Sodium hypochlorite under nondenaturing conditions. The Job’s

were

plot indicated that drug binds to the native and modified BSAs with 1:1

Fluorescence data indicated that 1:1 binding of drugs to bovine serum

stoichiometry. Binding constants underwent 11% increase and 73%

albumin (BSA) is associated with quenching of albumin intrinsic

decrease upon citraconylation and oxidation, respectively. Additionally,

fluorescence. The Job’s plot also confirmed that drug binds to BSA via

stability of oxidized and citraconylated BSA showed 50% decrease and

mentioned

30% increase, respectively. Thermodynamic analyses of the binding

thermodynamic parameters indicated that intermolecular interactions

process suggested that the major forces involved in the interaction of

between drug and albumin may change upon protein modification. The

furosemide to native and oxidized BSA are hydrophobic, whereas drug

theoretical analyses also suggested some conformational changes of

mainly binds to citraconylated form v iahydrogen bonds and van der

interacting side chains in subdomain IIA binding site (at the vicinity of

waals interactions. Changes in the protein surface hydrophobicity

W237), which were in good agreement with experimental data.

(PSH), were also investigated. We will discuss the importance of PSH

Decrease of protein surface hydrophobicity (PSH) was also observed

and stability in citraconylated/oxidized BSA and their effects on the

upon both albumin modification and drug binding.

investigated

by

using

stoichiometry.

various

Analysis

of

spectroscopic

the

methods.

quenching

and

furosemide binding characteristics. Keywords: Protein Hydrophobicity, Bovine Serum Albumin, Binding Keywords: Fluorescence

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Bovine Serum Quenching,

Albumin, Furosemide, Protein

Surface

Binding Site, Hydrophobicity,

Site, Glycation.

Journal of the Iranian Chemical Society, Vol. 9, Suppl. 1, June 2012

Abstract No.6

Abstract No.5 Increasing of Thermostability and Autolysis Resistance in a

Study of Interactions Between Antimicrobial Peptide LAH4

Neutral Protease by Site-Directed Mutagenesis

and Model Membranes

Seyedeh Akram Shirdel 1, Hamid Reza Karbalaei Heidari 2,

Matin Eslami 1, Faramarz Mehrnejad 1, Majid Taghdir* 2,

Khosro Khajeh*

1

Mahmoud Khadem-Maaref 3

1. Dept. of Biochemistry and Biophysics, Faculty of Biological Science,

1. Dept. of Cellular and Molecular Biology, Faculty of Science,

Tarbiat Modares University, Tehran, IR

Azarbaijan University of Tarbiat Moallem, Tabriz, IR

2. Dept. of Biology, College of Sciences, Shiraz University, Shiraz, IR

2. Dept. of Biology, Faculty of Sciences, University of Guilan, Rasht, IR


3. Dept. of Physics, Faculty of Science, Azarbaijan University of Tarbiat

Neutral proteases (NPs) are sensitive to high temperatures and


Moallem, Tabriz, IR experience functional and structural alterations upon heating. There are members of a family of homologous metalloproteases which differ

The designed histidine-rich amphipathic peptide LAH4 exhibits potent

considerably in thermostability and autolysis by amino acid differences

antimicrobial and DNA transfection activities. This peptide also was

at the protein surface. The rate-limiting step in thermal inactivation of

found to mediate intracellular delivery of protein-based vaccines to

NPs comprises local unfolding process that cause the enzyme to be

generate enhanced CD8+ T cell immune responses and antitumor

susceptible to autolysis as a result of exposing specific regions of its

effects, that all of them require interactions with cellular membranes.

structure,

some

Bilayer association of peptide has been shown to be strongly pH-

representative mutants in order to elucidate the thermal stability

dependent, with in-planar alignments under acidic conditions and

differences between wild type and mutant proteins, concerning point

transmembrane orientations when the histidines are discharged. The

mutations. The wild type enzyme was used in this study isneutral

mechanism by which these AMPs selectively attack the bacterial

protease from Salinivibrio Proteolyticus. The variants were cloned in

membrane is believed to depend on differences in membrane lipid

pQE-80L as expression vector. Maximum expression was reached at

composition. Herein, our results show that the difference in binding

30˚ C, 1 mM IPTG in LB medium. After purification of the enzyme with

affinity of LAH4 for palmitoyl-oleoyl-phosphatidyl-glycerol (POPG) and

Q-Sepharose column chromatography, the variants were characterized

palmitoyl-oleoyl-phosphatidyl-choline

for their kinetic and thermodynamic parameters, resistance to

molecular dynamics simulations. These results inform us of the

temperature and autolysis. Results show that our new mutations are

detailed location and orientation of the peptide with respect to the

able to slightly increase thermal stability while reduce proteolytic

bilayer as well as present that, the different binding affinity be related

cleavage. It was also concluded that there is no drastic changes in

to polar, electrostatic, and hydrophobic protein-lipid interactions that

thermodynamic parameters upon mutations. Far-UV CD and intrinsic

provide an understanding of what occur during membrane insertion.

fluorescence spectroscopyas

secondary and tertiary conformational

MD Simulations show that the peptide should have stronger

probes were also used to monitor the effect of mutations on the

interactions with anionic bilayer head group when compared to

structure of proteins.

zwitterionic bilayer head group. The results also indicat changes in the

Keywords: Autolysis, Local Unfolding, Thermal Stability, Variants And

According to our MD results and previous NMR experimental studies,

Spectroscopy.

LAH4 is more effective at the anionic bilayer comparing zwitterionic

critical

for

autolysis.

Hence,

we

constructed

(POPC)

by

using

all-atom

lipid acyl chain order when LAH4 is bound to the different membranes.

membrane in acidic condition.This results can be an effective means to improve antimicrobial properties and an approach in against of the growing health problem of antibiotic resistant and serve as an important foundation for future clinical applications to improve protein/peptide-based vaccine potency. Keywords: Antimicrobial Peptide, Drug Delivery, Molecular Dynamics Simulation, Membrane.

A3


Abstract No.7 Abstract No.8 The Stability Study of Recombinant Human Granulocyte-Colony-Stimulating Factor

Study on the Interaction of Lysozyme whit SDS and SOS by Fluorescence Spectroscopy

Narges Maleksabet* 1, Asghar Taheri-Kafrani 2, Mohammad Reza Masoumian 1

Sadegh Farhadian* 1, Behzad Shareghi 1, Masoud Salavati-Niasari 2, Nayere Bahamin 1, Somayeh Asgari 1, Parisa Nooraei 1

1. Department of Sscience and Biotechnology, Malek Ashtar University of Technology, Tehran, IR

1. Shahrekord University, IR

2. Department of Biotechnology, Faculty of Advanced Science and

2. Kashan University, IR

Technology, University of Isfahan, IR


(E-mail: [email protected]) Growth and differentiation of various blood cell lines from progenitor stem cells are regulated by a group of proteins known as hematopoietins including granulocyte-colony-stimulating factor (GCSF). G-CSF is a 19.6 kDa glycoprotein consisting of 174 amino acid residues.

G-CSF,

produced

mainly

by

macrophages,

induces

proliferation of neutrophil colonies and differentiation of precursor cells to neutrophils, and stimulates the activity of mature neutrophils. Recombinant human granulocyte-colony stimulating factor (rh-G-CSF) is a therapeutic unglycolysated protein is produced in E.coli in our institute and used primarily to reduce incidence and duration of severe neutropenia and its associated. The stability of recombinant proteins has become an increasingly important consideration as more protein therapeutics are developed.In this study, the purified rh-G-CSF was characterized by following the changes on the structure, purity, dimerization and aggregations of protein in time of 0-3 months and two temperatures (4 and 25 ℃) by using biochemical techniques including reverse-phase (RPC), size-exclusion chromatography (SEC), electrophoresis and circular dichroism. The results were compared with Neupogen filgrastim as reference standard. The purity of samples and the stability of the proteins against aggregations were measured by SEC and SDS-PAGE electrophoresis. According to the inspection chromatogram, obtained peak conforms to molecular weight of rh-GCSF without any aggregation forms in the protein structure (less than 1%) and disulfide bonds are in correct position. RPC results showed the similar hydrophobicity for rh-G-CSF and reference standard. CD results showed the same secondary and tertiary structure of G-CSF and reference standard in addition to the fact that the G-CSF secondary structure is predominantly helical. The obtained results approved that the rh-G-CSF was highly pure and comparable with the innovator products, Neupogen filgrastim and has a proper thermal stability in 4 and 25 ℃ in period of 3 months after production. Keywords: Rh-G-CSF, Stability, SDS-PAGE Electrophoresis, Circular Dichroism, Chromatography.

A4

Lysozyme, also called muramidase, a small monomeric globular protein first discovered by Alexander Fleming in 1922, is a basic protein belongs to the class of enzymes that lyse the cell walls of bacteria by hydrolyzing

the

bond

between

N-acetylmuramic

acid

and

N-

acetylglucosamine of the peptidoglycan. Lysozyme is antimicrobial protein widely distributed in various biological fluids and tissues including avian egg and animal secretions, human milk, tears, saliva, airway secretions, and secreted by polymorphonuclear leukocytes. In this paper, we designed to examine the effect of sodium dodecyl sulfate (SDS) and sodium octyl sulfate (SOS) on the solution structure of lysozyme using fluorescence and UV/Vis at different temperatures and pHs. All studies were carried out in quartz cells containing 0.1mM lysozyme and different Concentration SDS and SOS. The fluorescence spectrum of each suspension was measured, where the excitation wavelength was at 280 nm and the emission wavelength was between 290 and 450 nm (using 5 and 3 nm of slit width). The interaction of (SDS and SOS) with lysozyme was investigated by fluorescence spectroscopic techniques under physiological conditions. The results revealed that SDS and SOS caused the fluorescence quenching of lysozyme through a static quenching procedure. It was reported that 80% of lysozyme fluorescence was due to Trp62 and Trp108, and the oxidation of either Trp62 or Trp108 was accompanied by a drastic decrease in fluorescence intensity. In addition, Trp62 emission was in a higher proportion than that of Trp108 when lysozyme molecule was excited at 280 nm. Therefore it was reasonably proposed that SDS and SOS quenched the fluorescence of both Trp62 and Trp108 residues of lysozyme. Keywords: Lysozyme, Sodium Dodecyl Sulfate, Sodium Octyl Sulfate (SOS), Protein.


Abstract No.10

Abstract No.9 The Effect of pH on Recombinant C-Terminal Domain of

Docking of Cladribine and Fingolimod to P53

Botulinum Neurotoxin Type E (rBoNT/E-HCC)

and RAS exons of DNA

Mosayeb Rostamian 1, Seyed Jafar Mousavy* 1, Firouz Ebrahimi 1,

Azade Hoghoughi, Karim Mahnam*, Hajar Taheri,

Mohammad Reza Dayer 2, Ali Akbar Moosavi–Movahedi 3

Mehrdad Hoghoughi, M Mokhtarianpour

1. Departmet of Biology, Faculty of Science, Imam Hussein University,

Department of Biology, University of Shahrekord, Chaharmahal

Tehran, IR

Bakhtiari, IR

2. Department of Biology, Faculty of Science, Shahid Chamran


University of Ahvaz, Ahvaz, IR 3. Institute of Biochemistry and Biophysics, University of Tehran,

Some of the newest drugs that are effective on treating Multiple

Tehran, IR

Sclerosis disease (MS) are Fingolimod and Cladribine, but these drugs


are carcinogenic. It seems that due to aromatic rings, these drugs are able to bind DNA and can cause cancer by altering DNA structure.

Clostrdium botulinum neurotoxins cause botulism syndrome. One of

Drugs can bind to DNA by covalent and non- covalent bonds, and may

the recombinant vaccines against these neurotoxins is constructed

cause thermodynamic instability or damage on structure of DNA. These

using 93 residues of C-terminal domain of botulinum neurotoxin type E

changes

(rBoNT/E-HCC). In this report, we made an effort to investigate the

Understanding the effect of drugs from the point of structural and

effect of different pH on protein structure to assess if rBoNT/E-HCC

mechanical characteristics on the DNA is an important aspect in

could be used as a vaccine for oral administration. Initially, E.coli BL21

designing new drugs. Nowadays, docking is essential for designing of

(DE3) containing the synthetic gene of rBoNT/E-HCC were cultured

new drugs. Fingolimod and Cladribine were docked on four exons of

can

cause

abnormal

cell

division

cycle

or

tumor.

and the expressed protein, rBoNT/E-HCC,was purified by affinity

P53 and RAS genes that theire sequence mentioned in table 1 by

chromatography. Structural changes of rBoNT/E-HCC in the presence

Autodock 4 software. Routine methods and default parameters were

of different pH (2, 5, 7.4 and 9) were done by various techniques

used for docking procedure. Results of docking indicate that Van der

including circular dichroism (CD), fluorescence, aggregation, thermal

Waals interactions are greater than electrostatic interaction in

denaturation and UV-Vis spectroscopy. The aggregation experiments in

connection of these drugs to DNA, in all four sequences. Then these

the presence and absence of DTT indicated that rBoNT/E-HCC at pH 2

drugs are attached to DNA through hydrophobic interactions. These

is more resistant to thermal aggregation in contrast to higher pH 9.

two drugs were bond to DNA through minor groove. Data indicate that

Fluorescence experiments showed the more compact and more stable

binding of Fingolimod to DNA is stronger than Cladribine, thus it

structure for rBoNT/E-HCC at pH 2, and loosely folded structure at pH

appears that its carcinogenesis effect is greater. Results show

9. Thermal denaturation of protein indicated that the melting

Cladribine and Fingolimod can bind to exon one of RAS gene more

temperature was increased with increasing of pH from 2 to 9 up to

powerful than other exons.

2°C. This increment was caused by stabilization effects of increasing amounts of secondary structures which was caused by pH increments.

Keywords: Docking, Cladribine, Fingolimod, DNA.

We hypothesize that this finding is not in contrary with aggregation results, because, the nature of forces acting in aggregation and denaturation are not exactly the same. According to our findings we

Abstract No.11

advise further studies of protein from immunological point of view as an oral vaccine.

Structural and Allergenicity Properties of Recombinant Wildtype and Cys121Ser Mutant β-lactoglobulin

Keywords: Botulinum Neurotoxin Type E, Ph, Fluorescence, Circular Dichroism, Aggregation, Thermal Studies.

Asghar Taheri-Kafrani* 1, Abdol-Khalegh Bordbar 2, Thomas Haertle,3 1. Department of Biotechnology, Faculty of Advanced Science and Technology, University of Isfahan, Isfahan, IR

A5


2. Laboratory of Biophysical Chemistry, Department of Chemistry,

Depatment of Biology, University of Shahrekord, IR

University of Isfahan, Isfahan, IR


3. FIP, BIA, Institut National de la Recherche Agronomique, BP 7162744316 Nantes, FR

The binding of surfactant to protein is a widespread phenomenon and


plays a particularly important role in the activity of an enzyme. In this study the turbidimetric assay (activity) of lysozyme followed by the

β-Lactoglobulin (β-Lg) is a lipocalin, which is the major whey protein of

decreased optical density (OD) of a turbid cell suspension (about .3

cow milk and the milk of other mammals. However, it is absent from

mg/ml Micrococcus lysodeikticus) photometrically at 450 nm. Effect of

milk of primates. This globular protein of about 18 kDa is folded

sodium dodecyl sulfate (SDS) as an anionic surfactant on lysozyme

forming a b-barrel (or calyx) structure. Each monomer contains two

enzyme was investigated by UV-Vis spectrophotometry at pH 7.25 at

disulphide bonds and one cysteine at position 121 (Cys121). This free

35°c using sodium phosphate buffer. Measurements were carried out

thiol plays an important role in the heat-induced aggregation of β-Lg,

using 6×10-8 M concentration of lysozyme and a range of SDS solution

and, possibly, in the maintenance of its conformational stability. β-Lg is

concentration between 0.4 and 0.8 mM. It was found that by

one of major allergens in bovine milk. In this study, the expression in

increasing of SDS concentration, the rate of Micrococcus lysodeikticus

the yeast Pichia pastoris of a mutant bovine β-Lg, in which Cys121 was

lyses will be decreased. Vmaxvalue will be reduced by increasing of

changed into Ser

Analysis of

SDS concentration. Lysozyme consists of two domains: a α-domain

recombinant proteins by mass spectrometry has confirmed their purity,

with helical structure and a β-domain with predominantly β-sheet,

matching the calculated molecular mass with their mass theoretical,

separated by the active cleft. The cleft between the two domains

and the lack of post-translational modifications. Circular dichroism (CD)

includes the binding site for the substrate. Probably the contents of α-

and high performance liquid chromatography (HPLC) experiments

helix decreased sharply with the increase of concentration of SDS.

(Cys121Ser) was accomplished.

showed that the recombinant wild-type (WT) and Cys121Ser mutant retain native-like fold. The far- and near-UV CD spectra of WT and

Keywords: Lysozyme, Sodium Dodecyl Sulfate, Spectrophotometry,

Cys121Ser were very similar to that of the standard, indicating a

Activity, Protein.

similar secondary and tertiary structure. The mutation on the position 121 in amino acid chain completely blocks the irreversible aggregation induced by heat treatment according to electrophoresis results.

Abstract No.13

Compared to the recombinant wild-type protein, the mutant is less stable to temperature and disulphide reducing agents, and is much

Effects of Sodium Selenate on the Structure and Activity

more sensitive to peptic digestion. Binding of IgE from patients with

of Acetylcholinesterase

cow milk allergy to native β-Lg, wild-type β-Lg and Cys121Ser mutant β-Lg was aslo measured by ELISA. The calculated IC50 values for

Ezzatollah Fathi 1, Raheleh Farahzadi* 2

native and recombinant proteins were almost the same and the difference was not significant, indicating that the recognition of β-Lg by IgE from CMA patients is not impaired by recombinant WT and Cys121Ser mutant β-Lgs.

1. Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, IR 2. Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR

Keywords: β-Lactoglobulin, Allergy, Recombinant Protein, Circular


Dichroism, ELISA test. Acetylcholinestrase (AChE) (EC 3.1.1.7), is one of the most important enzymes in nervous system, and plays a role in the signal transduction in the somatic nervous system by termination of signal transduction in

Abstract No.12

the synapse. It has been reported that the function of this enzyme Kinetics and Spectrophotometric Studies on The Interaction

plays a role in Alzimer's disease. Selenium is one of the most important

of Sodium Dodecyl Sulfate With Chicken Egg White Lysozyme

micronutrient. Many investigations have been performed about the

in Aqueous Solution

physiological, biochemical and behavioral effects of this element, such as postponing the Alzheimer's symptoms in the elderly and delaying

Neda Zamani, Behzad Shareghi*

the initiation signs of skin aging. In this study the effects of different concentrations of Sodium selenate (0, 390, 870, 1300 µM) on AChE

A6


activity investigated using UV-visible spectroscopy, fluorescence

decreased enzyme activity and the main inactivation took place in 217

spectroscopy and circular dichroism

The results

Hz (p 1, showing anti-cooperativity between

Banafshe Rastegari*

substrate and inhibitor binding sites) and a KI value of 1.36 mM (better inhibitor relative to reference drug, acarbose ( KI =9.11±0.25 mM)).

Department of Biology, Faculty of sciences, Shiraz University, Shiraz, IR

From the result of this study it can be concluded that aloin can be


considered as a natural medication for the diabetes patient. Pectinases are biotechnologically important enzymes that involved in depolymerisation of the heterogeneous polysaccharide, pectin. A

A78


bacterium was isolated from fruit waste soil based on the potential

biotechnological industries therefore, methods improving the stability

degradation of the highly methylated pectin. According to the 16S

and functionality of HRP will clearly broaden the range of its present

rDNA sequence analysis, it was identified as Bacillus licheniformis BR1

and future applications. In the present study, chemical modification of

which produces an extracellular polymethyl galacturonase PMG (EC

HRP was carried out with 2,3-dichloromaleic anhydride and 2,3-

3.2.1.64) when cultured in medium containing citrus pectin0.1 %,

dimethylmaleic anhydride. Thermoinactivation, kinetic parameters and

peptone1%, yeast extract1%, pH 7.0. PMG production was optimized

activation energy of catalysis and structural changes of HRP upon the

(6.22 U/ml) and the enzyme was purified to homogeneity in two steps

modification were studied using spectroscopic techniques. The results

column chromatography. The enzyme was a dimeric protein and

indicated that 2,3-dichloromaleic anhydride increases thermal stability

exhibited an apparent molecular mass of 104 kDa by gel filtration

of HRP but 2,3-dimethylmaleic is not a stabilizer modifier for HRP.

chromatography and 56 kDa while running on SDS-PAGE. The enzyme

Catalytic efficiency and activation energy did not change remarkably

exhibited maximum activity at 60 °C, pH 6 with 49% of total activity at

following reaction of the enzyme with the carboxylic anhydrides.

90 °C for 30 minutes and extends range of pH stability (5.0-9.0).

Fluoresnce measurements indicated that the intensity of protein

Kmand Vmax of the PMG were determined 0.066 µmol/min and

emission decreases slightly upon the modification with both modifiers.

2.51mg/ml respectively, using pectin as substrate. PMG activity

A decrease in the distance between the heme and the tryptophan

significantly enhanced in the presence of 5 mM Ca2+, Cu2+, Mg2+,

residue or decrease in compactness of the structure and therefore

Mn2+ and Co2+, although Hg2+ and Fe2+ served as strong inhibitor.

exposure of trp residue to solvent may lead to this change. On the

The enzyme was identified as a metal-dependent pectinase, which was

whole,

inhibited by 5 mM of EDTA and showed suitable stability in the

dimethylmaleic implies that hydrophilization of the protein surface

presence of 0.1% SDS and Tween 80. In overall, regarding to acidic

results in thermal stability of the protein.

comparing

the

effect

of

2,3-dichloromaleic

with

2,3-

properties and high operational stability of the purified pectinase, it seems that PMG can be an ideal functional substitute for applications in

Keywords: Horseradish Peroxidase, Thermal Stability, Chemical

fruit juice industry, especially in citrus fruits extraction and clarification.

Modification, Spectroscopy.

Keywords: Pectinase, Bacillus licheniformis, Purification, Polymethyl galacturonase, Thermostablity. Abstract No.186 Design and Construction of Antagonistic VEGF Variant for Abstract No.185

Inhibition of Angiogenesis

Effect of Chemical Modification on Thermal Stability, Structure

Fahimeh Ghavami 1, Shirin Shahangian 1, Reza H. Sajedi* 2,

and Function of Horseradish Peroxidase

Shahriar Arab 3, Mahmoodreza Aghamaali 1, Kamran Mansoori 4

Rasool Nowruzi, Leila Hasani*

1. Department. of Biology, Faculty of Science, University of Guilan,

Department of Biological sciences, Institute for Advanced Studies in

2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat

Rasht, IR Basic Sciences (IASBS), Zanjan, IR

Modares University, Tehran, IR


3. Department of Biophysics, Faculty of Science, Tarbiat Modarres University, Tehran, IR

Biocatalysts are increasingly employed in chemical processing because


of their selectivity as well as their potential as a greener alternative to


chemical catalysis but, they are not usually tolerant to the presence of


organic solvents, extremes of pH or high temperatures. Consequently, there is great interest in developing strategies to improve protein

Because of its central role in pathological angiogenesis, vascular

stability in desired reaction media. Compared with the strategies for

endothelial growth factor (VEGF) has become a major target for anti-

obtaining stable enzymes, chemical modification is a simple and

angiogenic

effective technique. Horseradish peroxidase (HRP) has achieved a

heterodimeric antagonistic VEGF variant (HD-VEGF). In this antagonist,

prominent

binding domains for the VEGF-receptor KDR/Flk-1 is present at one

position

in

the

pharmaceutical,

chemical,

and

therapies.

We

report

here

the

construction

of

a

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pole of the dimer, whereas the other pole carries domain swap

nanoparticles in brain is mediated by induction of oxidative stress in

mutations, which prevent binding to receptor. As HD-VEGF can only

microglia cells and astrocytes. However neuronal presynaptic terminals

bind to monomeric receptors, it does not lead to signal transduction.

could be other targets for nanoparticles due to high turn over of

Thus, HD-VEGF blocks KDR-mediated VEGF activities that are crucial in

synaptic vesicles in these terminals. In this case it has been shown that

the angiogenic process and is therefore a promising, multipotent

ferritin, as an iron nanoparticle, inhibits glutamate uptake. Glutamate is

compound in the treatment of angiogenesis-related diseases. The

the major excitatory neurotransmitter in mammalian central nervous

receptor binding domain of wild type VEGF with N-terminal His-tag was

system. Excessive extracellular concentrations of this neurotransmitter

expressed as inclusion body in E. coli and refolded successfully in our

could cause hyper activation of glutamate receptors and result in cell

lab.To construct the heterodimeric VEGF variant, the precise KDR

death. In the present work the effect of ZnO and CuO, two metal oxide

binding sites were determined from the crystal structure of VEGF-KDR

nanoparticles that are widely used in industry, has been studied on the

complex (PDB ID: 3V2A). Considering to the φ and ψ dihedral angles,

extracellular concentration of glutamate in rat Synaptosomes. We

the distance between the first and the last amino acids and the length

found that ZnO and CuO nanoparticles can cause remarkable increase

of these segments, some sequence with equall length and distance

in extracellular glutamate in Synaptosomes. We suppose that

compared to those of VEGF were searched in pdb site. The binding

glutamate uptake is inhibited in the presence of these metal oxide

sites in VEGF crystal structure were replaced by suitable sequences.

nanoparticles.

The model of designed mutant VEGF was compared to native one after energy minimization process. Based on constructed model, the

Keywords: Synaptosomes, Neurodegenerative Diseases,

receptor binding domain of mutant VEGF gene was designed,

Nanoparticles.

ZnO/CuO

synthesized and inserted in pET-21a expression vector with additional N-terminal Strep-tag which providing a system that able to purification of

heterodimeric

from

homodimeric

variants

by

two

affinity

Abstract No.188

chromatography. The mutant VEGF was expressed in E. coli and after purification of heterodimeric variant, its anti angiogenic property will be

Determination of Microscopic Dissociation Constants of

investigated in future.

Tryptophan

Keywords: Angiogenesis, Antagonistic VEGF, Heterodimer, Strep-tag.

Marjan Farhangi*, Mohammad Reza Housaindokht, Narjes Ashraf Ferdowsi University of Mashhad, Mashhad, IR (E-mail: [email protected])

Abstract No.187 ZnO and CuO Nanoparticles Increase Extracellular Glutamate

The study of the interactions between surfactants and proteins is of significant

Concentration in Synaptosomes of Rat Brain

scientific

macroconstant

pKa

and values

technological are

importance

commonly

known

[1].

The

while

the

Shahrbanoo Rafiei Taghanaki, Gholamhossein Riazi*,

microconstant values (especially tautomerization constant Kz) are often

Shahin Ahmadian

not recognized [2]. The concept of microscopic constants is important and interesting, and it has the advantage of being directly related to

Department of Biochemistry, Institute of Biochemistry and Biophysics,

the specific functional groups involved, thereby, enabling investigators

University of Tehran, Tehran, IR

to interpret such parameters in terms of molecular structure.


Determination of microscopic constants and tautomeric ratios has played an important part in understanding the ionic composition of

In modern life, human beings are exposed to nanoparticles from

many biologically active molecules, particularly because all proteins fall

ambient air and certain work places. There have been increasing

into this class. Furthermore, the microscopic constants and tautomeric

reports that inhaled nanoparticles can reach the brain through two

ratio describe the amount of various species as a function of pH [3].

main routes: blood circulation and olfactory bulb penetration. More

The present work is an attempt to study the effect of anionic

over exposure to nanoparticles in vivo increases the risk of

surfactant SDS on the dissociation equilibria of the amino acid

neurodegenerative

that

tryptophan. In this study, we have determined macroconstant Ka1,

nanoparticles can kill neurons. How ever the mechanisms underlying

Ka2 values, tautomerization constant kz and microconstants k11, k12,

diseases

while

in

vitro

studies

show

this phenomenon are still unclear. It is believed that the toxicity of

A80


k21, k22 values for the Tryptophan aqueous solution and tryptophan

Abstract No.190

solutions in the presence of different concentrations of SDS. Monoclonal Antibodies in the Treatment of many Diseases in Keywords: Microscopic Dissociation Constants, Tautomeric Ratios,

Mice Experimentally Using Veterinary Clinic of Sina Gilan,Iran

Tryptophan, Sodium Dodecyl Sulfate.

Mojtaba Norouzi* 1,Mohammad Norouzi 2, Naser Ghaemi 3, Nour Amirmozafari 4 Abstract No.189 1. Department of Microbiology, Islamic Azad University Lahijan, IR Probing Crucial Amino Acids Role in Chondroitinase ABCI

2. Department of Microbiology, Islamic Azad University Rasht, Doctor

Enzyme Activity by Site-directed Mutagenesis

Veterinary Medicine (DVM), IR 3. Department of Biotechnology, Tehran, IR

Shima Hatamzadeh 1, Khosro Khajeh* 2, Majid Sadeghizadeh 2,

4. Department of Microbiology, Tehran, IR

Abolfazl Golestani 3


1. Department of Biology , faculty of science , University of Guilan,

Monoclonal antibodies(MAB)have found increasing use experimental

Rasht, IR

therapies.great limitation of their use is that they are recognized by the

2. Department of Genetics,Faculty of Biological sciences,Tarbiat

patient as being of foreign origin and an antiglobulin response is


provoked.recombinant DNA technology offers the ability to convert

3. Department of Clinical Biochemisty, School of Meicine, Medical

these rodent antibodies into a more human form.there are currently

Sciences University of Tehran, Tehran, IR

several different strategies which can


humanized antibodies resulting in different degrees of humanization

be adopted to generate

can be achieved ranging from chimeric antibodies with a combination Chondroitinase ABC I is an112.5kDa enzyme with 997 amino acid

of human constant regions with rodent variable regions to fully

.This enzyme is a GalAG (galactosaminoglycan) depolymerizing lyase

reshaped antibodies where the variable regions are also humanized.At

with a broad-specificity and depolymerizes a variety of GAG

present the available data on clinical use of chimeric and reshaped

substrates,including C4S (chondroitin 4-sulphate), DS (dermatan

antibodies is very limited. The rat IgG2b antibody CAMPATH-1G has

sulphate), C6S (chondroitin 6-sulphate) and hyaluronic acid. Previous

been shown to be both useful as an immunosuppressive antibody as

studies suggested that cABC1 enzyme promote neural system

well as in the treatment of lymphoid malignancies . The reshaped

regeneration by degrading GAG chains followed by decreasing CSPG s

version, CAMPATH-1H, was

inhibitory effect so there is a wide range of application of the enzyme

malignant cells from the blood and bone marrow in two patients with

in medicine .cloning and expression of the enzyme have been done

B-cell lymphoma . A more sustained course of the human antibody

successfully in BL-21. Three Dimensional structure analysis with x-ray

(126 mg over 30 days and 86 mg over 43 days) was tolerated than

crystallography suggested that the enzyme has three major domains.

had previously been used for the rat antibody.In the future in

The optimum pH and temperature in Tris buffer is pH=8 and 37 ˚C,

veterinary clinic of Sina gilan, it is clear that a majority of monoclonal

respectively . In order to increase thermal stability we applied quik

antibodies produced for therapy will be humanised for the reasons

change mutagenesis. We use a thermophile strain thetaiotaomicron

discussed above. As far as improvements in the abilities of these

WAL2926 as a template to guide us the best selection of amino

antibodies to interact with human effector mechanisms goes it seems

acids for enzyme engineering. In this study, proline 485 has been

that there is unlikely to be any major differences between chimeric and

substituted with alanine. finally the activity and stability of the wild

fully reshaped antibodies.important about humanised antibodies is

type and its varint has been discussed and proven.

whether there are any sequences contained within the variable region

successfully used to clear detectable

frameworks, complimentary determining regions or constant region Keywords: Chondroitinase ABC I, Galactosaminoglycan, Site Directed

allotypes which can be processed and presented as T-cell epitopes.

Mutagenesis Stability. Keywords: Monoclonal Antibody, Chimeric Antibody, Recombinant DNA Technology, Humanized Antibody, Antiglobulin.

A81


Department of Physics, Institute for Advanced Studies in Basic

Abstract No.191

Sciences (IASBS), Zanjan, IR Ocular Delivery of Lysozyme Through Soft Contact Lenses


Farkhondeh Khanjani*, Fatemeh Javadi, Mohamad Taheri,

Polymer translocation is the passage of a polymer through a narrow

Reyhaneh Sariri

channel in a wall. This phenomenon is very ubiquitous in biological environments, for example the passage of proteins through the inner-

Department of Biology, University of Guilan, Rasht, IR

and outer-cellular membranes. In addition, recently, it has found


applications in the fast and cheap sequencing of nucleic acids [D. Branton et al, Nature Biotech. 26, 1146 (2008)]. Polymer entry into the

The aim of this work was to study the effect of pH and temperature

nano-channels is important in the optimization of the polymer

on the absorption and release of the lysozyme through soft contact

translocation [M. Wanunu et al, Nature Nanotech. 5, 160 (2010)] and

lenses to specify optimum condition for this drug delivery to the eye.

the polymer separation [J. Han et al, Phys. Rev. Lett. 83, 1688

Lysozyme is a part of the innate immune system. This drug has

(1999)].Here, we study the polymer entry time into an asymmetric

antibacterial property and it's utilized to disinfect the eye. This property

nano-channel similar to the alpha-hemolysin protein channel, under

is useful particularly for the people who use soft contact lenses to

the electric field effect, using simulations. We show that the existence

repair their eye s’ refractive defects. In this work Contact lenses were

of a wider part before the narrow channel reduces the polymer entry

made by polyacrylamide hydrogel. Lysozyme was added to the lenses

time into the channel, dramatically. Moreover, we have performed

in two ways. In one method the drug was loaded into the lenses by

simulations of polymer translocation through a channel with the

soaking the lenses in lysozyme-phosphate buffer solution and in

dimensions of the alpha-hemolysin channel [N. Nikoofard and H. Fazli,

another method, the drug was added into the gel during its

Phys. Rev. E 85, 021804 (2012)]. The order of the entry times and

polymerization. Then contact lens released therapeutic levels of drug in

their ratio from the two sides are close to the related experiment [S. E.

a fresh phosphate buffer solution for a few days. The absorption and

Henrickson et al, Phys. Rev. Lett. 85, 3057 (2000)].Before entry to the

release of the lysozyme were measured in various conditions of pH and

channel, the polymer is compressed to the wall under the electric field.

temperature by UV-Vis spectrophotometer. Normalized lysozyme

So, we study the statics and dynamics of a polymer compressed to the

activity (substrate units per mg of enzyme) was determined by using

wall under the electric field, theoretically and with simulations. We

an assay that is based on the hydrolysis of the outer cell membrane of

compute the activation energy barrier of the polymer for entering the

Micrococcus lysodeikticus. Samples of native and hydrogel-released

channel and its attempt time for crossing the barrier. Our theory for

lysozyme were mixed with the M. lysodeikticus suspension in

the activation barrier can explain the results of the previous

phosphate buffer and the decrease in turbidity was measured. The

experiments on the polymer translocation through the alpha-hemolysin

activity of the hydrogel-released lysozyme was compared with native

channel

lysozyme to confirm functionality of the released protein. The results

communications) 83, 050801 (2011)].

[N.

Nikoofard

and

H.

Fazli,

Phys.

Rev.

E

(Rapid

showed that the released lysozyme activity level was essentially identical to the native lysozyme. Therefore, lysozyme functionality was

Keywords:

not affected upon incorporation and release through the hydrogels.

Activation Barrier.

Polymer

Translocation,

Asymmetric

Nano-Channels,

Keywords: Soft contact lens, Controlled drug release, Lysozyme, Ocular delivery.

Abstract No.193 Design new Peptide as TrkB inhibitor

Abstract No.192

Marzieh Kafshdozi Amin* Polymer Entry Into an Asymmetric Channel Under the Electric Field Effect

Qazvin University of Medical Sciences,Qazvin, IR (E-mail: [email protected])

Narges Nikoofard*, Hossein Fazli Cancer is one of the most killer disease in the world. Cancer pathology is dependent to many proteins such as Neurotrophins family such as

A82


NGF, BDNF,NT3/4,NT3 and their receptors displayed crucial roles as

data

proliferation, migration, differentiation, survival, apoptosis, In previous

pentamine. Results showed that in the presence of tetraethylen

indicated

studies improved TRK B is as oncogene agent and BDNF bind to trk b

pentamine reduced apparent Km and Vmax i.e. tetraethylen pentamine

and active signaling angiogenesis for tumor proliferation.In the current

with Ki=0.1 mM inhibits the enzyme by linear mixed inhibitory effect.

century using intelligent biomolecule such as antibody and peptides is

In linear mixed inhibition, the inhibitor can bind to the enzyme at the

hopeful therapies in cancers.In this study we design of new peptides

same time as the substrate. However, the binding of the inhibitor

by using monte carlo methods and study the effect of them on cancer

affects the binding of the substrate, and vice versa. This type of

cell lines. For this aim we use backrub protocol and docking with trkB

inhibition

as receptor. our result showed that this protocol could improve the

concentrations of substrate.

can

considerable

be

reduced,

inhibitory

but

not

effects

for

overcome

by

tetraethylen

increasing

peptide design for cancer treatment. At the first step of this protocol Chickpea,

Copper-Containing

we designed peptide library by using sequence tolerance method in

Keywords:

rosetta3.3 package,then peptide energy optimization performed by

Tetraethylen Pentamine, Linear Mixed.

Amine

Oxidases,

backrub protocol for finding peptides with more stability,the five of best peptides selected based on R software and peptide 3D-structure prediction performed by using molecular dynamic in Hyperchem 7 software. the final step is Docking of peptides with receptor trkb in

Abstract No.195

HADDOCK then cyclotraxin and designed peptides. Synthesis of Cyclooxygenase Inhibitor 2-(1-benzyl-alkyl thioKeywords: TrkB inhibitor, Cancer Treatment, Peptide Desining, Rosetta3.3 Package.

5-imidazolyl)-3-pheenyl-1, 3-Thiazolidin-4-one as Anticancer Agent

Narges Shahini* 1, Farzin Hadizadeh 2 1. School of Pharmacy,Mazandaran University of Medical Science,

Abstract No.194

Mazandaran, IR A Study on the Interaction of Chickpea Seedling Copper Diamine Oxidases by Tetraethylen Pentamine

2. School of Pharmacy, Mashhad University of Medical Sciences, Mashhad , IR (E-mail: [email protected])

Sona Talaye* 1, Asadollah Asadi 1, Mojtaba Amani 2 Recently study was shown that enzyme cyclooxygenase have a role in 1. Dept. of Biology, Faculty of Science, University of Mohaghegh

cancer tissue. Since the derivatives of thiazolidine 4-one and other

Ardabili, Ardabil, IR

pharmacophore patterns of central ring have a cox-2 inhibitory effect,

2. Dept. of Science, Faculty of Medicine, University of Medical science,

thus we decide to synthesis of novel derivatives of thiazolidin -4-ones.

Ardabil, IR

At first, benzyl amine hydrochloride was produced. Then the mixture


of, dihydroxy acetone, thiocianate potassium was reacting for 72 hours. By alkylation and oxidation formyl imidazole was obtained .The

Copper amine oxidase are soluble dimeric enzymes, each monomer

resultant aldehyde was reacted with aniline and thioglicolic in two ways

contains one Cu(II) ion and one organic prosthetic group 2,4,5-

including classic (in one and two step ways) and microwave method

trihydroxyphenylalanine quinone as cofactors. Inhibitors represent

until the title compounds were obtained.1-Classic method: In the one

important role in the study of catalytic properties of copper amine

step way ,all the reactant (resultant aldehyde, aniline and thioglicolic)

oxidase and they also find a wide application in physiological research.

were refluxed in dean stark apparatus in dry toluene for 48 hours.B: In

They catalyze the oxidative deamination of primary amines to

the two step way, at first aldehyde and aniline react in dean stark for

aldehydes with a ping-pong mechanism consisting of a transamination,

24 hours to give imine intermediate, then thioglicolic acid was added

followed by the transfer of two electrons to molecular oxygen which is

and reacted for 24 hours. In two stages: at first aldehyde and aniline

reduced to H2O2. Kinetic parameters Km and Vmax of purified enzyme by

react in dean stark for 24 hours after synthesizing dimidiate product,

analysis of Lineweaver - Burk plot was determined 3.3 mM and 0.95

thioglicolicacid was added and react for 24 hours.2- Microwave

mmol/min/mg, respectively. In this study interaction chickpea diamino

method: All of reactant was treated at 800 w, 100 c for 15 minute. TLC

oxidase withtetraethylen pentamine was studied. Analysis of kinetic

was used to evaluate the progress of the reaction and purify material.

A83


Structure elucidation was done using NMR and IR spectrum.Yields of

Abstract No.197

production of thiazolidone in both method is less than previous steps (80-90%).the yield of two stage method was about 10% more than

Dynamics and Flexibility of Mnemiopsin in

one step method and microwave only decreased the time of reaction

the Presence of Calcium

the yield was unchanged.As the reaction is reversible, the water that is produced during the synthesis, takes the reaction to left side thus

Shima Tarahomi 1, Reza Hassan Sajedi* 2, Bijan Ranjbar 2, Majid Taghdir 1

without using the dean stark and drying solvent, the yield decrease.

1. Dept. of Biology, Faculty of Sciences, University of Guilan, Rasht, IR Keywords: Cyclooxygenase Inhibitor, Derivatives Of Thiazolidine 4-

2. Dept. of Biochemistry, Faculty of Biological Sciences, Tarbiat

One, Anti Cancer.

Modares University, Tehran, IR (E-mail: [email protected]) Mnemiopsin is a photoprotein that belongs to the EF-hand calciumbinding proteins (EFCaBP). Ca2+ is involved in specific and selective

Abstract No.196

regulation

of

cellular processes.

Many

of

these

functions

are

Study on p53R2(small Subunit of Ribonucleotide Reductase)

accomplished through the interaction of Ca2+ with specific proteins.

Function Via Exploring its Expression Level After Different

EFCaBPs have eminent properties which change upon interacting with

Doses of Irradiation in Human Mouth Epitelial ,KB, Cells

calcium ions. It is generally believed that Ca2+ confers on proteins an increased thermal stability, affect flexibility and rigidity, interface

Farideh Habibi, Reza Rofugaran,

reversible unfolding, protein-protein interaction and etc. Our previous

Bahram Goliaei*

spectroscopic (CD and fluorescence) studies indicate that the apo-form (Ca2+-depleted) of the protein adopt a closed conformation when

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IR

compared to the Ca2+-loaded once. In this study, flexibility and dynamics


of mnemiopsin were investigated for apo-mnemiopsin and Ca2+-loadedmnemiopsin with fluorescence acrylamide quenching and limited

Ribonucleotide reductase ,(RNR), is the enzyme that provides

proteolysis. Calcium is removed by TCA (trichloroacetic acid) precipitation

deoxyribonucleoside triphosphates (dNTP) for DNA synthesis. The

to obtain apo-mnemiopsin. Fluorescence quenching was carried out by

recently discovered p53-inducible small subunit of ribonucleotide

the addition of 1M acrylamide to protein solutions at an excitation

reductase

wavelength of 295 nm and an emission wavelength of 337 nm in a

(p53R2)

mitochondrial

DNA

plays

essential

synthesis.

It

roles is

in

DNA

believed

repair to

and

provide

Perkin-Elmer luminescence spectrometer.

Limited proteolysis was

deoxyribonucleotides for DNA repair since the protein is expressed via

performed by incubation of protein with thermolysin at 37 ˚C.

p53 which is involved in DNA damage checkpoints. Because of late

Acrylamide quenching of tryptophan fluorescence is widely used to

expression of p53R2 (12h) in response to DNA damage to repair, it

monitor tryptophan environments in proteins. Dynamic quenching

was suggested that this protein has other functions in the cell.Taken

analysis revealed that Ca2+-loded- mnemiopsin was quenched more than

together, in this study, we are investigating the expression level

the apo-form. Molecular modeling indicates microenvironment of Trp21 is

changes of p53R2 after different doses of irradiation treatment of KB

important to explain this experimental data. Limited proteolysis

cells. This project hopefully will be able to shed light on the function of

experiments can be used to identify the sites of enhanced flexibility or of

p53R2 in DNA repair and mitochondrial DNA synthesis. We did KB cell

local unfolding of a polypeptide chain. Although thermolysin has been

culture, and obtained doubling time (20.5 h) and clonogenic assay for

classified as proteases with broad specificity, it should be considered that

obtaining plating efficiency (38%) to investigate cell survival. In final

thermolysin cleaves mostly the amino side of bulky and hydrophobic

step, will do western blotting to probe the protein level in different

amino acids. Mnemiopsin proteolysis results indicate that Ca2+-loaded

doses of x-ray. The detailed results will be discussed.

form is more sensitive to proteolysis than apo-form. Therefore, the present results from limited proteolysis and digestion experiments as well

Keywords: RNR, p53R2, DNA repair, Radiation.

as quenching analysis can be explained by the fact that Ca2+-loadedmnemiopsin is more flexible in some regions when compared to apomnemiopsin. Keywords: Mnemiopsin, Flexibility, Quenching, Proteolysis.

A84


Department of Chemistry, Faculty of Science, Ferdowsi University of

Abstract No.198

Mashhad, Mashhad, IR Mobile Phone Electromagnetic Field Effect on HEK Cell Line


Trough Luciferase Reporter Some of Iron( III) Salen Complexeshave a very desirable Anti-Tumor

Yahya Sefidbakht* 1, Saman Hosseinkhani 2,

activity against MCF7 cells. Their Anti-Tumor activity is the result of

Masoud Torkzadeh-Mahani 2, Iman Tavakkolnia 3, Reza Faraji-Dana 3,

optimizing a collection of descriptors. Quantitative structure activity

Ali Akbar Saboury 1, Ali Akbar Moosavi-Movahedi 1

relationship (QSAR) of the Anti-Tumor activity of Fe(III)-salen and salen-like complexes was studied .The method of DFT (B3LYP/Lanl2dz)

1. Institute of Biochemistry and Biophysics, University of Tehran, IR

was used in order to optimizing 3D shape of the complexes. A pool of

2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat

descriptors was calculated, 1497theoretical descriptors by Dragon


software and quantum-chemical parameter, shielding NMR, electronic

3. School of Electrical and Computer Engineering, University of Tehran,

descriptors by Gaussian 09 and AIM software. Study of structure and

Tehran, IR

activity relationship was performed with multiple linear regression


(MLR) and Artificial Neural Network (ANN). In nonlinear method, the Adaptive Neuro-Fuzzy Interference System (ANFIS) was used to select

Wireless technology and mobile phones are now inseparable aspect of

the most effective descriptors. The ANN-ANFIS model with high

life. The possibility of adverse effects of these fields is subject of

statistical significance (R2train= 0.99, RMSE = 0.138, Q2LOO= 0.82)

variety of researches. The cellular effects of electromagnetic field are

has better capability to predict Anti-Tumor activity of new compounds

still no clear. There is variety of targets for EMF action on cell from the

series of this family. Based on this study antitumor activity of this

cell membrane to the genomic content and regulatory systems. Hence,

compound is mainly dependent on the geometrical parameters and

it is valuable to have a sensor that can report the EMF effects from

position and nature of the substituent of the salen ligand.

inside the cell. Luciferase enzyme is well known as reporter enzyme. Luciferase was transfected into HEK293T cell line by Lentiviral vector.

Keywords: QSAR, Iron (III) Salen Complexeshave, Artificial Neural

We have studied the effect of 940 MHz Waveguide EMF on HEK293T

Network, Multiple Linear Regression.

cell line for 10- 90 minute in 2.5 cm plate at 37°C. At the indicated time points, the cells were lysed by CCLR buffer and luciferase activity was measured. The results show that within 30 minutes the luciferase

Abstract No.200

activity decreases down to 20% while after 1 hour interestingly the exposed group activity was 20% higher than control one. This shows

Diminishing the Allergenicity of Beta Lactoglobulin by Digester

that the cell response can compensate the first 30 min activity lose in

Copper Complexes

next 30 min exposure. Therefore, at overall after 1 hour the exposed cells are 20% more active than control ones. This shows the

Sajedeh Ebrahim Damavandi* 1, Adeleh Divsalar 2, Ali Akbar Saboury 1

mechanism in HEK293T cell line that can resist the damage caused by the applied EMF. The reason for the 40% remediation between 30 min


to 60 min exposure time might be due to the activation of cell

2. Dept. of Biological Sciences, Tarbiat Moallem University, Tehran, IR

response via heat-shock proteins.


Keywords: Mobile Phone, Electromagnetic Field, HEK cell Line,

Beta-lactoglobulin (BLG) is one of the bovine milk proteins that causes

Luciferase Reporter.

allergic response. The cleavage

of

the allergic sequence is the

superlative method for decreasing protein allergenicity. As regarding to the literatures, Cu complexes make the proteins slicing to peptides. In this research, four new Cu complexes were synthesized and

Abstract No.199

characterized. These Cu complexes are:[Cu bpy Cl2] complex 1, [Cu QSAR Study on Fe (III)-salen-like Complexes as Potent

(bpy )2 Cl2] complex 2, [Cu (dien) OH2] (NO3-] complex 3, [Cu(trien)

Anticancer Agents

(NO3)2complex 4, which supposed to reduce protein allergenicity through cleaving BLG, as mentioned above. New copper complexes

Zahra Ghanbari*, Mohammad reza Housaindokht

were incubated with the protein. After 30 hours of incubation (as

A85


optimum time), the samples were studied by SDS-PAGE, fluorescamine

domain

and ELISA methods. the effect of Cu complexes on protein digestibility

concentrations, nearly similar to that of the intact VEGF. Because of

was proved by SDS-PAGE and fluorescamine methods. Also ELISA

the key role of VEGF in wound healing, some experiments to elucidate

method admitted that protein degradation cause allergenicity to be

the effect of the refolded receptor binding domain on this process, as

decreased. It seems that Cu complexes are able to operate as artificial

well as some other angiogenic assays are under way.

of

VEGF

showed

high

angiogenic

potential

at

low

proteases and cure related diseases by degradation of target protein . Keywords: VEGF Receptor Binding Domain, Refolding, Angiogenesis, Keywords: Beta-Lactoglobulin, Allergenicity, Cu Complexes.

HUVEC Proliferation Assay, Wound Healing.

Abstract No.201

Abstract No.202

Expression, Refolding and Angiogenic Activity of

Comparison of APX activity of Different Cultivars of the Maize

Human VEGF Receptor Binding Domain

in Cold Stress Condition

Shirin Shahangian 1, Reza H. Sajedi* 2, Kamran Mansouri 3,

Saber Zahri, Huorieh Aliakbari Majd*, Mahdi Razavi, Saeid Latifi Navid

Sadegh Hasannia 2, Shirin Jalili 2 Department of Biology, Faculty of Sciences, University of Mohaghegh 1. Department of Biology, Faculty of Science, University of Guilan,


Rasht, IR


2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR

One of the consequences of environmental stress on plant is an

3. Medical biology research center, Kermanshah University of Medical

increase in the cellular concentration of reactive oxygen speaice, which


are subsequently converted to hydrogen peroxide . ROS demage cell


components such as DNA , protein and lipid ,… . plants have two type of antioxidative systems against ROS , nonenzymatic and enzymatic

Of the multitude of growth factors that regulate physiological and

system.APX is an important part of the enzymatice antioxidative

pathological angiogenesis, vascular endothelial growth factor (VEGF) is

system that control the peroxides such as hydrogen peroxide

believed to be the most important. VEGF is an endothelial cell-specific

concentration in cell , in reaction APX using ascorbate as a substrate ,

mitogen and an angiogenic inducer as well as a mediator of vascular

and catalys transfer of electrons from ascorbate to peroxide ,

permeability. VEGF is essential for developmental angiogenesis and is

producing dehydroascorbate and water as products. We analyzed the

also required for female reproductive functions, endochondral bone

activity of APX in different cultivers of maize to cold stress. These

formation, fracture and wound healing, neuroprotection after ischemia

cultivar includes chilling sensitive and chilling tolerance cultivars, APX

or spinal cord injuries. VEGF actions are mediated through binding to

activity determined with assay containing hydrogen peroxide and one

two receptor tyrosine kinases, VEGFR-1 and VEGFR-2. VEGF binds to

of the reducing substrate , such as ascorbate . for ascorbate the

its receptors via its receptor binding domain. There are many inter and

activity was followed as the decreas in A290 due to the consumption of

intra subunit disulfide bridges in the VEGF receptor binding domain. In

ascorbate in an assay containing 10mµ hydrogen peroxide and

this study, the receptor binding domain of VEGF was overexpressed as

0/5mµascorbate in buffer. Result indicated APX activity Increase later

inclusion bodies in E. coli and refolded by multi-step refolding

cold strees ,and APX activity were much higher in the chilling tolerance

procedure. Considering to the fact that VEGF homodimerization is

maize than chilling sensitive maize.

essential for its receptor binding and biological activity, we followed dimerization of the protein in the refolding process. Based on reducing

Keywords:

and non-reducing SDS-PAGE, the refolded protein was present

Ascorbate, Hydrogen Peroxide.

primarily in the dimeric form with little amount of monomer. Circular dichroism and fluorescence spectroscopy studies confirmed the correct refolding of this domain. The angiogenic potency of this variant of VEGF was also investigated using human umbilical vein endothelial cells (HUVEC) proliferation assay. Interestingly, the receptor binding

A86

Reactive

Oxygen

Speaice,

APX,

Dehydroascorbate,


Keywords: Gold Nanorods, Surface Plasmon Resonance, Circular

Abstract No.203

Dichroism. Circular Dichroism Study of DNA-Gold Nanorod Nanobioconjugates Abstract No.204

Azadeh Azizi 1, Bijan Ranjbar* 1,2, Tahereh Tohidi Moghadam 2, Zahra Karami 1

Synergic Antioxidant Activity of Tea (Camellia Sinensis) and Mentha Pulegium

1. Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR

Rohoollaeh Razmgar, Reyhaneh Sariri*, Mohamad Taheri,

2. Department of Nanobiotechnology, Faculty of Biological Sciences,

Rezvan Shahmohammadi

Tarbiat Modares University, Tehran, IR (E-mail: [email protected])

Department of Biology, Faculty of Science, University of Guilan, Namjoo Street, Rasht, IR

Nobel plasmonic nanoparticles have offered variety of applications in biomedicine

and

biosensing.

Nanobioconjugates

of

DNA


and

nanoparticles have been introduced in designing new generation of

Active oxygen species (ROS), produced during some natural aerobic

nanoprobes for detection purposes. Amongst plasmonic nanoparticles,

processes, could be involved in various disorders. Harmful action of

gold nanostructures of rod morphology have been nominated as good

free radicals can be reduced by antioxiant compounds that scavenge

candidates for different biological and biochemical applications. So far,

free radicals and detoxify the organism. Antioxidants are recognized for

there have been a number of reports based on drug/gene delivery and

their potential in promoting health and lowering the risk of many

diagnostic capability of gold nanoparticles. However, fundamentals of

human disorders such as cancer, hypertension and heart disease.The

interaction between nanoparticles and biomolecules are yet to be

aim of this study was to investigate the antioxidant activity of various

understood. In some cases, conjugation of biomolecules with

mixtures of tea and Mentha Pulegium.In practice, 80% methanol

nanoparticles of interest might induce adverse effects on the structure

extracts of individual samples of dried tea leaves and Mentha Pulegium

and function of

the biomolecules, where the applicability of

were prepared. Different ratios of extracts were then made for

nanoparticles remains under question. Herein, we present the

synergistic studied.Total phenolic content (TPC) and antioxidant

interaction between gold nanorods and a single-strand DNA, to monitor

activity of various mixtures of tea-menthe pulegium were measured

the conformational changes of nucleic acid upon interaction with rod-

using Folin-Ciocalteau, DPPH-free radical scavenging and ferric-

shaped nanostructure. Colloidal gold nanorods were synthesized

reducing antioxidant power assay (FRAP). The correlation of TPC with

according to conventional sequential seed mediated growth method.

DPPH and FRAP assay of plants extracts were investigated.According

Formation of rod shape was characterized by monitoring the typical

to the results, the ratio of 50 - 50 showed the highest antioxidant

surface plasmon resonance absorption bands in the visible and near

power relative to isolated samples of tea and Mentha pulegium.

infrared

by

Considering that both plants had high antioxidant activity, flavin

transmission electron microscopy (TEM) and Atomic absorption

bridges are likely to be higher in synergistic mixture, and enhance

spectroscopy (AAS), to reveal the shape and size. The purified samples

antioxidant power properties of poly-phenolic hydrogen peroxide.

region.

The

nanorods

were

further

characterized

were then interacted with a fixed concentration of nucleic acid (0.5 mg. mL-1) and incubated at ambient temperature for several hours.

Keywords: Antioxidant, Synergic, Tea, Mentha Polegium, Extract,

Circular dichroism spectra of the complexes were recorded in the UV

Total phenolic content, DPPH radical Scavenging Assay, Ferric –

region (200-300 nm). Results showed that typical bonds of ssDNA at

Reducing Antioxidant Power Assay.

245 and 268 nm have not changed considerably and the nucleic acid has maintained its conformation upon interaction with gold nanorods. Outcomes of this investigation could encourage the possibility of using gold

nanorods

in

the

upcoming

research

fields

of

applied

nanobiotechnology for detection, immobilization and stabilization of nucleic acids.

A87


photoproteins such as aequorin and obelin and there is no information

Abstract No.205

about the mechanism of cetenophorephotoroteins and architecture of Spectrofluorimetric Study of the Interaction of

its

coelentrazine

binding

site.

In

native

aequorin

and

obelinphotoproteins, exists a water molecule (W1) in the binding

Daunomycin Antibiotic with Histone H1

pocketthat makes hydrogen bonds with the phenolic OH of the 2-ρ-

Seyed Jalal Zargar*

hydroxybenzyl, the Oγ-atom of Thr166, and the carbonyl oxygen of Ile105. The main role of W1 is considered to be the stabilization of

Department of Cell and Molecular Biology, School of Biology, College of

coelenterazine moiety. In the mnemiopsin, W1 that exists in the

Science, University of Tehran, Tehran, IR

binding pocket of native aequorin and obelin is missing and the


hydroxyl group of Ser128 establishes a hydrogen bond with 2-phydroxybenzyl one hand and the other to with Ile124 and thus possibly

Histone H1 is a very lysine rich histone fraction of chromatin which

creating a network of hydrogen bondsin this position.In the well-known

appears to play an important role in the compaction and stability of the

photoproteins, Ile105 and Thr166 are essential in the formation this

structure of chromatin . Daunomycin is an antitumor anthracycline

network and their corresponding residues are Ile124 and Val183 in

antibiotic wildly used as a chemotherapentic agent for the treatment of

mnemiopsin. Therefore, V183T and S128G substitutions based on

various cancers.In the present study, we have investigated the

hydrogen bond network around the ring of coelentrazine were

interaction of daunomycin with the purified calf thymus histone H1 in

performed. On the contrary, the V183T and S128G mutants showed

solution using fluorescence spectroscopy technique in 20 mM

75% and 19% activity respectively when compared to wild type

phosphate buffer , pH=7.0 and 1 mM EDTA at room temperature. The

variant.V183T mutant was active in higher concentrations of calcium

results show that daunomycin decreases the fluorescence emission of

and its maximum wavelength of bioluminescence spectrum had about

histone H1 at 325 nm and induces hypochromicity at the emission

20 nm blue shift relative to the wild type (470 nm vs. 490 nm). Other

spectrum of this protein. Also the fluorescence emission of daunomycin

characteristics of the mutant did not show any changes. CD and

is increased at 595 nm and hyperchromicity is induced at the emission

fluorescence spectroscopyandmolecular modelingstudiesshowed some

spectrum of this drug after incubating with histone H1.Increasing ionic

structural changes in the mutants. Based on homology modeling

strength elevates this effect. The results suggest that H1 can be

studies, thishydrogen bond network has been removedin S128G

considered as a target for daunomycin action at the chromatin level.

mutant, while in V183T mutant, an additional hydrogen bond was observed. The results indicated that the hydrogen bond network

Chromatin,

Keywords:

Histone

H1,

Daunomycin,

Fluorescence

Spectroscopy.

around the ρ-hydroxybenzyl ring in position C2 of coelenterazineis important in the bioluminescence activityof photoproteins. Keywords: Site-directed mutagenesis, Photoprotein, Mnemiopsin, Coelenterazine.

Abstract No.206 Role of Hydrogen Bond Network Around the p-hydroxybenzylringof Coelenterazine in

Abstract No.207

Mnemiopsinbioluminescence Activity by Site-directed Mutagenesis

Stabilization of Glucose Oxidase Upon Immobilization on Albumin Amyloid Nano-fibers

Zohreh Jahani 1, Maryam Molakarimi 1, Reza H. Sajedi* 2, Saman Hosseinkhani 2, Majid Taghdir 2

Sara Farahi 1, Mehran Habibi-Rezaei* 1, Ali Akbar Moosavi-Movahedi

2

1. Dept. of Biology, Faculty of Sciences, University of Guilan, Rasht, IR

1. School of Biology, College of Science, University of Tehran, Tehran, IR


2. Institutes of Biochemistry and Biophysics, University of Tehran, IR



(E-mail: [email protected]) Glucose oxidase (GOD, EC 1.1.3.4) is an oxidoreductase with Photoproteins are Ca2+-regulated proteins that emit light. So far, the

numerous biomedical and industrial applications. It catalyzes oxidation

most investigations of photoproteins have been done on coelenterate

of glucose to gluconic acid. Molecular oxygen accepts electrons to

A88


produce hydrogen peroxide upon the recation by GOD. Hydrogen

model which contains enough structural details of F-actins to study the

peroxide is produces gradually and does its antimicrobial activity in

mechanism of this attraction. For this purpose, we apply the 4-sphere

effective and gentle manner. GOD also is routinely used to evaluate

model for the structure of G-actin. In this model, G-actin is composed

glucose in physiological fluid included with medical diagnosing kits.

of four subdomains, one of which carries the most charge of G-actin

Measurement of glucose concentration is important for determination

(sd1). Using this 4-sphere model, we consider real structure and size

of blood glucose levels in diabetic patients. GOD has been successfully

of F-actin in addition to bending and twist rigidity and helical charge

immobilized onto various scaffolds to improve its stability. In this study

distribution of F-actins.Applying MD simulation to a group of these F-

we used covalent binding strategy for immobilization of GOD on

actins, we observe that they attract each and form a hexagonal lattice

glycation induced biocompatible amyloid nanofibers of bovine serum

of the same lattice size as the experiment results. Also it is observed at

albumin in which glutaraldehyde was used as a cross-linker. The

equilibrium that counter-ions tend to assemble midline between two

optimum concentration of the enzyme for immobilization was

neighbor F-actins. This distribution of counter-ions around F-actins and

determined at 160 µg GOD per mg amyloid nano-fibers. . The kinetic

how sd1s arrange on F-actins will shed light on understanding the

parameters of the free and immobilized GOD were also determined.

mechanism.Accurate look at equilibrium details of arrangement of sd1

Despite on a decreasing of the catalytic performance (kcat/Km) of the

of two neighbor F-actins and plotting the force on them along F-actins,

enzyme upon immobilization, the covalently bound GOD on amyloid

it is concluded that at equilibrium, F-actins will rotate and twist until

nano-fibers retained almost 40-70% of its activity after incubation at

they expose their sd1s close to each other. Because of their high

temperatures 25, 45 and 65ºC but a major decrease in the activity was

negative charge, they will attract a cloud of counter-ions in a region

occurred for free enzyme under the same conditions. Hence, presented

between themselves. This cationic cloud will locally attract the two F-

activity in a broad range of temperature most importantly at 40°C< in

actins in its side and it is effectively seen that the two F-actins attract

immobilized form compared to the free enzyme are explained.

each other.

Interestingly, comparison between pH profiles of the free and immobilized enzymes indicates on increasing pH sensitivity of the GOD

Keywords: F-actin, Bundle, Counter-ion, Multivalent salt, 4-sphere

activity in the pH range lower than 6 and the optimum pH shift from 5

model, MD simulation.

to 6 upon immobilization was observed. Keywords:

Catalytic

Nano-Fibers,

Amyloid,

Glucose

oxidase,

Abstract No.209

Glycation, Immobilization. Amyloid Peptide Nanofibrils: from Structure to their Lipid Peroxidative Effects Abstract No.208

Maryam Ferdousi* 1, Mehran Habibi-Rezaei 1, Saeed Balalaei 2, Salt-induced Bundle Formation of F-actin Using a Detailed

Ali Akbar Moosavi-Movahedi 3

Model 1. School of Biology, University College of Sciences, University of Tehran,

Sarah Mohammadinejad* 1, Ramin Golestanian 2, Hossein Fazli 1

Tehran, IR 2. Department of Chemistry - K.N.Toosi University of Technology, IR

1. Department of Physics, Institute for Advanced Studies in Basic

3. Institute of Biochemistry & Biophysics (IBB), University of Tehran, IR

Sciences (IASBS), Zanjan, IR


2. Rudolf Peierls Centre for Theoretical Physics, University of Oxford, Oxford, OX1 3NP, UK, GB

Alzheimer’s disease is a neurodegenerative disorder which is the most


common cause of senile dementia. Amyloid beta (Aβ) is an amphiphilic peptide of 39 to 43 amino acids which is produced from a

Actin filaments assemble into networks or bundles helped by linker

transmembrane precursor by a proteolytic cleavage, furthermore it is

proteins, depending on their different roles in cell function. Similar

the main component of the neuritic plaques of the Alzheimer’s

patterns have also been observed experimentally in solutions of F-actin

disease.In this research some of the structural, physicochemical, and

rods with multivalent salt. In bundle structure, F-actins place close to

cytotoxic properties of the Aβ (25-35) and one of its modified, cys-

each other in parallel formation. So despite their same high negative

amyloid beta (25-35), which has an additional cysteine residue on its

charge, why do they attract each other?Here we aim to represent a

N-terminal, have been investigated. The objective of this research is to

A89


comprise these two peptides and understand the effects of the

assay.Gel electrophoresis results indicated that 1%, 0.5%, 0.1% and

presence of cysteine residue on the rate of amyloid oligomer and

0.05%

nanofibril

physicochemical

nanoparticle. MTT assay indicated that the average viability of cells

properties of the oligomers and nanofibrils have been investigated. As

treated with chitosan/plasmid nanoparticles was about 97% versus

a result, α helix to β sheet transition, degree of β-aggregation and

80% for Lipofectamine 2000. Average complex size of18 and 50KD

morphology of non-modified and modified Aβ peptides were studied

chitosan

using

extrinsic

respectively.Protection of nucleic acid in the serum is a major problem

fluorescence and microscopic methods including TEM and AFM,

in gene therapy that could be solved by chitosan for its Strong

respectively. As a result, fibrilogenesis of cys-amylid beta (25-35)

attachmentto DNA.Furthermore using chitosan nanoparticles as a gene

showed the same rate as it was for Aβ (25-35). Also, their lipid

delivery system is a safer way of gene transfection for its lower

peroxidative effects on model liposomal membrane are reported.

cytotoxic effect.

production.

Moreover,

spectropolarimetery

Amyloid

Keywords:

structural and

(CD),

Beta,

thioflavin

Nanofibrils,

T

(ThT)

Alzheimer’s

Disease,

concentrations

molecular

weight

form

were

chitosan/

197

and

pTracer-CMV2

299

nm

Keywords: Chitosan Nanoparticle, Cytotoxicity, Lipofectamine, T47D

Aggregation.

cell line.

Abstract No.210

Abstract No.211

Effect of Chitosan Nanoparticles on T47D Viability

could

Interaction of Human Umbilical Cord Derived Stem Cells with Biodegradable PLLA Scaffold

1

2

Elham Malakooty poor* , Nematollah Gheibi , Mohamadreza Baghaban Eslaminejad 3, Shahrokh Safarian 4, Fatemeh Bagheri

Asadollah Asadi* 1, Fariba Mansourizadeh 2, Shahrbanoo Oryan 2

3

1. Biology Department, Faculty of science, University of Mohaghegh 1. Department of Medical Biotechnology, Qazvin University of Medical


Sciences, Qazvin, IR

2. Biology Department, Faculty of science, University of Tarbiat

2. Department of Physiology and Medical Physics, Qazvin University of

Moallem Tehran, Tehran, IR

Medical Sciences, Qazvin, IR


3. Department of Stem Cells and Developmental Biology, Cell Sciences Research Center, Royan Institute for Stem Cell Biology and

Tissue Engineering (TE) is the regeneration of biological tissues

Technology, ACECR, Tehran, IR

through the use of cells, with the aid of supporting structures and

4. Department of Cell and Molecular Biology, School of Biology

biomolecules. Mimicking architecture of extracellular matrix is one of

University College of Science, University of Tehran, Tehran, IR

the challenges for TE. The ideal scaffolds provide a framework and


initial support for the cells to attach, proliferate and differentiate, and form an extracellular matrix (ECM). Electrospined Poly-L-lactic acid

This study describes the low cytotoxicity of chitosan/DNA complexes on

(PLLA) was selected for this study. They haven’t immunologic response

T47D in compare with Lipofectamine as a novel way of gene

and have FDA permission for medical use. Scaffold surface topography,

transfection.The chitosan/DNA nanoparticles were synthesized through

chemical microstructure and mechanical properties have been shown

the complex coacervation method of the chitosan solution with

to significantly influence cell behaviors such as adhesion, growth and

pTracer-CMV2 plasmid. In this regard two diffrent molecular weight of

differentiation. The umbilical cords derived stem cell interaction with

chitosan(18-50 KD) and several concentrations of each including 1%-

(PLLA) scaffold via evaluation of cell adhesion to synthetic nanofibrous

0.5%-0.1%-0.05%-0.01%-0.005%-0.001% were used. samples were

polymeric scaffold. During the experiment, human mesenchymal stem

run through an agarose gel to examine the synthesis of complexes of

cells (MSCs) were successfully isolated from the umbilical cords and

nanoparticles. In order to measure the Particle size and zeta potential

cultured in the PLLA scaffold and then the viability and proliferation of

of nanoparticles we used zetasizer. T47D cell line treated with

the cells determined via both of trypan blue exclusion test and MTT

chitosan/plasmid nano particle complex synthesized using above-

assay. Results exhibited high biocompatibility which verified by no

mentioned dilutions of chitosan. Treatment with Lipofectamine2000

significant difference between the number of the cultured cells on the

was taken as the control. The Cell viability was determined by MTT

scaffold and control samples. Furthermore cell morphology, adhesion

A90


and growth capability were investigated by scanning electron

Abstract No.213

microscopy (SEM( and finally fluorescence staining with DAPI (4', 6diamidino-2-phenylindole) used for evaluation of mesenchymal cells

Effect of Inorganic Arsenic on the Expression Level of GST

adhesion and penetration into the scaffolds nanopores. These

Metabolizing Enzymes

observations enabled us to conclude that the cell culture on appropriate

scaffolds

helps

the

MSC

expansion

growth

Iraj Saadat*, Mostafa Saadat

and

differentiation.

Department of Biology, College of Sciences, Shiraz University, Shiraz, IR (E-mail: [email protected])

Keywords: Tissue Engineering, Cell Interaction, PLLA Scaffold, Cell Viability, Proliferation, Adhesion.

Millions of people around the world drink water with elevated concentrations of arsenic. Epidemiological studies have showed that chronic exposure to arsenic is associated with several diseases

Abstract No.212

including skin, lung and bladder cancers as well as vascular diseases

Study of some Chemical Compounds on Tyrosinase

and diabetes. While several studies showed that arsenic is a human

Inhibition and Their Potential Application in Melanoma

carcinogen, the mechanism underlying of this toxicity remain largely unknown. One possible mechanism involves the induction of oxidative

Nematollah Gheibi*

stress via the generation of reactive oxygen species (ROS). Glutathione and related enzymes are involved in cellular protection against ROS.

Department of Biophysics, Qazvin University of Medical Sciences,

Since Glutathione S-transferases (GSTs) involve in the metabolism of

Qazvin, IR

inorganic arsenic, the aim of the present study was to demonstrate the


expression level of GSTM1 (a member of GST class mu), GSTT1 (a member of GSTclass theta) and GSTO2 (a member of GST class

Tyrosinase, an enzyme that catalyzes the rate limiting step in melanin

omega) genes in HeLa cell line in the response of sodium arsenite.. In

biosynthesis and found abundantly in melanoma, considered as a

order to determine whether the GSTM1, GSTT1 and GSTO2 mRNA are

molecular therapeutic target for development of novel prodrugs for

transcriptionally regulated by sodium arsenite, HeLa cells treated at the

selective treatment of melanoma. In addition, tyrosinase inhibitors are

final concentration 2µM sodium arsenite for 24 h. The expression level

becoming important constituents of cosmetic products in relation to

of GSTM1, GSTT1 and GSTO2 was determined with the quantitative

hyperpigmentation. The effects of some aromatic and fatty acids on

real time PCR. The expression of GSTs metabolizing enzymes in

activity of tyrosinase were assessed by kinetic studies. Caffeic, p-

sodium arsenite treated cells was slightly decreased compared to

comaric acids used as substrates in the catecholase and cresolase

untreated cells, but the alterations were not significant.

reactions of mushroom tyrosinase. 2-amino benzoic acid, 4-amino benzoic acid, nicotinic acid, picolinic acid and oleic acid showed

Keywords:

inhibitory activity on cresolase and catecholase reactions. The

arsenite.

GSTM1,

GSTT1, GSTO2,

Expression level,

Sodium

inhibition mode of nicotinic and picolinic acid were competitive in both activities of enzyme, and their inhibition constants (Ki) were determined as 1.21 and 1.97 mM for cresolase activity and 2.4 and

Abstract No.214

2.93 mM for catecholase activity, respectively. 2-aminobenzoic and 4aminobenzoic acids showed non-competitive inhibition for the two

Hemoglobin Fructation in the Presence of Iron-chelating

activities with Ki of 5.15 and 3.8 µM for cresolase activity and of 4.72

Drugs

and 20 µM for catecholase activity, respectively. Oleic acid showed mix manner of inhibition with Ki of 0.85 and 0.68 mM in cresolase and

Naghmeh Sattarahmady* 1,2, Hossein Heli 3

catecholase reactions, respectively. Although nicotinic, picolinic and oleic acids have significant cytotoxic effect against melanoma tumor cell line, such effect didn’t obtained with caffeic, p-comaric, 2-amino benzoic and 4-amino benzoic acids.

1. Department of Medical Physics and Engineering, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR 2. Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, IR

Keywords: Tyrosinase, Melanoma, Kinetic, Cytotoxicity, Acids.

3. Department of Chemistry, Science and Research Branch, Islamic Azad University, Fars, Shiraz, IR

A91



physiological pH. Two redox transitions were appeared in the voltammograms which were related to the redox processes of

Proteinopathies or protein conformational diseases such as Alzheimer’s,

CoII/CoIII and FeII/FeIII in the solid state of CoHCF. The modified

Parkinson’s diseases and type II diabetes, are conditions that arise

electrode was successfully applied to the electrooxidation of nitrite and

from the misfolding and aggregation of proteins in non-native

sulfite and these substrates were oxidized electrocatalytically on the

conformations. Non-enzymatic mechanisms such as glycation or

modified electrode surface via the active FeIII species. The catalytic

oxidation are a modifying factor that leads to proteinopathy, by

rate constants, the electron transfer coefficients and diffusion

affecting the structure and function of proteins which have essential

coefficients involved in the electrocatalytic oxidation of the compounds

role in diabetes. Identification of anti-glycation compounds is attracting

were

considerable interest.In this study, deferiprone, deferasirox and

amperometric determination of nitrite and sulfite.

reported.

The

modified

electrode

was

applied

to

the

desferal, three iron chelators used in the treatment of β-thalassemic patients, were chosen to explore their effects on the fructation of

Keywords:

hemoglobin. Our results indicated that deferasirox cannot prevent AGE

Electrocatalysis, Modified electrode, Nanoflowers, Nitrite, Sulfite.

Cobalt

hexacyanoferrate,

Cobalt

nanoflowers,

and carbonyl formation but it reduces the functional changes of hemoglobin, heme losses and helix depletion due to fructation. Deferiprone and desferal, on the other hand, prevent AGE formation

Abstract No.216

and inhibit changes in the structure and function of hemoglobin during the fructation process.

Measuring the Activity of Cytomegalovirus Promoter Using an in Vivo

Keywords: Glycation, Hemoglobin, Iron chelators, Proteinopathies.

Fateme Mortazavi* 1, Masood Torkzadeh 2, Saman Hosseinkhani 2, Mokhtar Jalali Javaran 2 Abstract No.215 1. Biotechnology Department, Kerman Graduate University of Nanoflowers of Cobalt: Synthesis, Characterization and

Technology and International Center for Science High Technology and

Application for the Electrocatalytic Oxidation and

Envirinmental Sciences Kerman, Kerman, IR

Determination of Sulfite and Nitrite

2. Biochemistry Department, Tarbiat Modares University, Tehran, IR (E-mail: [email protected])

Hossein Heli* 1, Iman Eskandari 2, Naghmeh Sattarahmady 3, Ali Akbar Moosavi-Movahedi 4

Transgenic technologies are dependent upon genetic tools among which sufficiently strong promoters for the construction of expression

1. Laboratory of Analytical and Physical Electrochemistry, Department

genes are very important. Reporter genes are essential for the

of Chemistry, Science and Research Branch, Islamic Azad University,

quantitative analysis of gene elements that potentially regulate gene

Fars, Shiraz, IR

expression. Several kinds of reporter genes have been developed and

2. Department of Chemistry, Islamic Azad University, Firouzabad

luciferase reporter gene is the most favored for functional analysis of

Branch, Firouzabad, IR

promoters and enhancers, due to rapid, sensitive and reproducible

3. Department of Medical Physics, Shiraz University of Medical Sciences

assay system.In this study we investigated the activity rate of

and Pharmaceutical Science Research Center, Shiraz University of

cytomegalovirus promoter of mammalian viruse in model plant cell

Medical Sciences, Shiraz., IR

suspension of Nicotiana tabacum via firefly luciferase. The sequence of

4. Institute of Biochemistry and Biophysics, University of Tehran,

firefly luciferase (codon optimized luciferase gene LUC+) from pgl3

Tehran, IR

control vector was introduced in to plox vector via appropriate primers


and two restriction enzymes (BamHɪ and Xhoɪ). The luciferase gene was cloned in Plox vector so cytomegalovirus promoter used to drive

Cobalt hexacyanoferrate (CoHCF) nanostructure was synthesized by

the expression of firefly luciferase in suspension cells of Nicotiana

anodic oxidation of metallic cobalt nanoflowers in a solution of

tabacum. The plox vector which contains luciferase reporter was

K3Fe(CN)6. The synthesized CoHCF sample was then employed to

transformed to cells. Measurement of luciferase activity was done in

prepare a modified carbon paste electrode. The modified electrode was

intact cells. Our result show that the cytomegalovirus promoter can be

characterized electrochemically in a phosphate buffer solution at

A92


recognized by plant RNA polymerase so we measured 14,000 RLU/sec

Padina Vaseqi maqvan, Reza Rasoolzadeh*

after 5 days of transfection. Science and Research Branch, Islamic Azad University, Tehran, IR Keywords: Cytomegalovirus

Promoter, Luciferase, Cloning, In vivo


assa. The isolation of a novel gene, TSGA10,is described by differential mRNA display which is expressed solely in adult human testis. It seems likely that the gene is expressed during spermatogenesis possibly in

Abstract No.217

spermatocytes. The gene is composed of 19 exons extending over QSAR Study of Neuraminidase Enzyme by Molecular Mechanic

more than 80 kb. The complete cDNA contains an open reading frame

Method, for Nano Drug Design

of 2094 nucleotides, which appears to encode a novel protein. It was predominantly expressed in the testis in adult normal tissues.Cancertestis genes are a group of genes expressed in testicular germinal cells

Reza Rasoolzadeh*, Maryam Mousavi

and a range of human cancers. Testis-specific gene A10 (TSGA10) is Science and Research Branch, Islamic Azad University, Tehran, IR

expressed in testis and actively dividing and fetal differentiating


tissues. Testis-specific gene antigen (TSGA10) is expressed in fetus, testis and frequently in human solid cancers and acute leukemias,

Nowadays drug design is made by two methods namely QSAR &

making it a candidate for immunotherapy. There is also evidence for

Docking.QSAR reveals a quantitative relation between structure &

potential TSGA10 involvement in cell proliferation.It was reported its

function based onHamt & Hansh equations. Statistical analysis

expression TSGA10 in human monocyte-derived dendritic cells (DC)

,molecular mechanics &molecular graphics have done a great

and macrophages in vitro and in murine spleen CD11c(+) cells ex vivo.

assistance in drug designing. For thepurpose of understanding of drug

It is proposed that TSGA10 could influence the function of antigen

designing methods we should have a complete knowledge of Receptor,

presenting cells (APC) via its interaction with cytoskeletal proteins such

Ligand, Binding site & Target site.Since H1N1 influenza A infection is

as vimentin.Autoimmune polyendocrine syndrome type 1 (APS1) is a

highly contagious ,its spread as epidemics& pandemics has made it a

rare monogenic autosomal recessive disorder.We used the methods

horrible disease.The WHO has many concerns inthis issue & expends

geometry optimization, Molecular Dynamics , Langevin Dynamics and

millions of dollars to produce drugs to suppress or treatthis disease.For

Monte carlo and The force fields are MM,AMBER,BIO(charmm) and

treatment of this disease a thorough knowledge of neuroaminidase

OPLS and temperatures are 295,300,305,310,315.By these methods

protein is essential in order to produce potent drugs to suppress this

were evaluated and significat results were obtaind.

enzyme. Due to virus's genomic inconstancy & point mutations, drugs that are no longer useful against this virus should not be used & new

Keywords: TSGA10 protein, TSGA10 gene, Molecular Dynamics,

more potent & suppressing drugs must be designed .We studied the

Geometry Optimization, Charmm.

drug

binding

sites

in

dielecterics(32,63&78,39)

varioustemperatures(310,315,329&333

K),

using

in

Bioinformatics

,molecular mechanics & MM+ Mont carlo methods.We measured the

Abstract No.219

potential energy of amino acids binding to the drug. Drugbinding sites are mor dependant to dielectric constants rather to temperature and the optimum dielectric constsnt is 39/78. Keywords:

Molecular

mechanic,

Nano Study and Simulation of the Na+/k+ Channels Proteins Membrane, Using MD/MM Methods

Influenza

A,

Dielectric,

Naghmeh Ezzati, Reza Rasoolzadeh*

Neuraminidase enzyme. Science and Research Branch, Islamic Azad University, Tehran, IR (E-mail: [email protected]) Abstract No.218 Sodium channels are integral membrane proteins the form ion Nano Theoretical Studies of Testis-Specific Protein/gene 10

channelsconducting sodium ions through a cell,s plasma membrane .

structure of Homo Sapiens and its Comparison with the

the voltagesensitivity of this channel is due to positive amino acids

TSGA10 protein/gene of Rattus Norvegicus

located of every third position voltage gated sodium channels have

A93


three types of states. channels are classified according to the type of

average amount of plasma glucose increases, the fraction of glycated

ion for which the channel is selective, e.g. K+ channels, Na+ channels,

hemoglobin increases in a predictable way. In this study, hemoglobin

Cl− channels. Voltage gated cation (Na , K ) channels are responsible

was incubated with doxorubicin and with different concentrations of

for the generation and propagation of action potential in neurological

glucose and was studied by uv-visible spectroscopy technique.

signal transmission . K1 selective channels are widely spread in all

Observations showed a hyperchromic effect upon Hb treatment by

organisms and have the ability to conduct K1 ions at near diffusion

doxorubicin, however this effect was reliefed through glycation in

limit. The KcsA channel shares sequence and structure similaritieswith

elevated concentrations of glucose.

all members of the K1 channel family.We have used geometry Hemoglobin,

optimization ,molecular mechanics simulations to calculate the relative

Keywords:

binding energies of Na and K in the KcsA ion channel .We investigated

Spectroscopy.

Doxorubicin,

Glycation,

Ultraviolet

the complex of na and k ions with ion channels in different tempreture (300, 305, 310, 315) and calculated potential energy of these molecule in force fields MM, AMBER, BIO (charmm) and OPLS. Maximom +

potential energy of comlex of Na

ion channels with MONTE CARLO

and OPLS method at T=300 K is 3614.84 kcal/mol .

Abstract No.221 Purification and Biochemical Characterization of Anacidic Metalloprotease from Serratia sp. HR-1

Keywords: Molecular mechanic, KcsA ion channel, OPLS, AMBER,

Hajar Rezanejad*

Hyperchem.

Department of Biology, Faculty of sciences, Shiraz University, Shiraz, IR (E-mail: [email protected]) Abstract No.220 Proteases are used in a great variety of commercial sectors.An acidic The Study of Glucose Interference on Interaction of

extracellular protease produced by anisolate from soilsamples was

Doxorubicin and Hemoglobin

purified to homogeneity in three steps including ammonium sulfate

Fatemeh Ebri Mehraban*, Seyed Jalal Zargar, Mehran Habibi-Rezaei

precipitation, SP-Sepharose cation-exchange and Sephacryl S-200 size exclusion chromatography. The purified protease exhibited an apparent molecular mass of 50 kDa either by SDS-PAGE or gel filtration

School of Biology, University College of Science, University of Tehran, IR

chromatography. It was found as a cold-adapted acidic protease with


optimumtemperature and pH of 30-35 oC and 4.0, respectively. The protease showed 24% of its initial activity after incubation at 50 oC for

Hemoglobin

transport

60 min, which indicates a relatively low thermostability. The enzyme

metalloprotein in the red blood cells. Quaternary structure and the

was classified as a metalloprotease according to its unaffected function

associated subunit interaction property of tetrameric Hb confers it the

in the presence of inhibitors: Aprotinin, E-64, Chymostatin, Pefabloc

regulatory property when small ligands such as metabolites or drugs

SC, Pepstatin, Leupeptin, Bestatinand Antipain-dihydrochloride and its

bind to it and modulate the functional activity of this important protein.

complete inhibition by 10 mM of EDTA. Althoughthe enzyme was

Protein-Drug interactions play an important role in a variety of

identified as a metalloprotease, no significant effect of 1,10-

biological processes and disease treatment.Doxorubicin (DOX) is a very

phenanthroline on caseinolytic activity showed Zi+2independency of

effective anti-tumor drug that is effective in the treatment of solid

the protein.

tumors. It has been suggested that on reduction DOX reacts directly

thermolysin-like metalloprotease inhibitor, revealed that the purified

with oxyHb. It seems the role that Hb plays in disposition of DOX or its

protease is not a member of M4 family of metallopeptidases. In overall,

interaction with the drug is important. The direct interaction of this

regarding tothe acidic properties and low temperature stability of the

protein with drugs may also affect its functionally important structural

protease, it proposed that the purified acidic metalloprotease can be

conformation.Binding of some ligands to Hb induce alterations in the

an ideal choice for applications in food industry especially in cheese

structure and function of this protein. However, competitive binding

making.

displayed

(Hb)

by

is

different

the

iron

ligands

containing

may

result

oxygen

from

No inhibitory function of Phosphoramidon, as a

allosteric

effects.Hemoglobin exposure to plasma glucosecauses glycation of

Keywords:

hemoglobin in a non-enzymatic glycationpathway. Normal levels of

Protease Inhibitors.

glucose produce a normal amount of glycated hemoglobin. As the

A94

Metalloprotease,

Serratiasp.HR-1,

Acidic

Protease,


1. Department of Chemistry, Imam khomeini International University,

Abstract No.222

Qazvin, IR pKa Calculation of the Positively Charged Amino Acids in a

2. Department of Biophysics, Qazvin University of Medical sciences,

Protein Chain, An Assessment of Theoretical Procedure

Qazvin, IR

Mohammad Izadyar*, Neda Zavvar, Mohammad Reza Hosseindokt

3. Department of Biochemistry and Genetic, Qazvin University of Medical sciences, Qazvin, IR (E-mail: [email protected])

Department of Chemistry, Faculty of sciences, Ferdowsi University of Mashhad, Mashhad, IR

The inhibitory effects of some flavonoids on the diphenolase activity of


mushroom

tyrosinase

have

been

investigated

with

the

In this study, the aqueous pKa for two cationic amino acids including

spectrophotometric technique. The results of kinetic assays showed

lysine and arginine in a peptide chain which composed of eight glycine

that the flavonoids induced a reversible inhibition on the enzyme

molecule have been calculated. HF and B3LYP methods using the 6-

activity. Furthermore, gallic acid and quercetin show non competitive-

31+G(d) [1-3] combined with solvation energies that were computed

type of inhibition. The chrysin and naringin induced a competitive

by the SCRF-(CPCM/UFF) continuum models as implemented in

manner of inhibiton. The inhibition constants have been determined for

Gaussian 09 computational package [4]. pKas were further calculated

these flavonoids and the inhibition strength follows the order of:

using two thermodynamic schemes (scheme1 and 2), namely the

quercetin < chrysin < naringin < gallic acid. The values of the inhibitor

direct method and the proton exchange method with the inclusion of

binding constant (KI) obtained 16.0, 7.9, 3.0, 1.5 mM, respectively.

an explicit solvent water molecule. The results of this research verify

Thus the flavonoids played an important role in the inhibition of the

that the direct method is not suitable for computing pKa of the amino

mushroom tyrosinase enzyme.

acids in a peptide chain, while the other scheme in the presence of water molecule significantly improved the pka in comparison to the

Keywords: Inhibition, Flavonoids, Kinetic, Mushroom Tyrosinase.

experimental data. The combination of the proton exchange scheme and CPCM-UFF model performed well with mean absolute deviations (MADs) of ~0.9 pKa unit. Because of the convergence problems, the

Abstract No.224

inclusion of large numbers of water molecule in scheme 2, the computation procedure in the solvated model produces weak results.

Nanobiosensors Designed to Detect Hydrogen Peroxide by

Comparison between the pKa α, β and random (R) structures of the

Using Catalase Polymer Nafiyn

studied proteins, reveals that the strongest acidic character belongs to β, α and R, respectively at the HF and B3LYP levels of the theory. The

Saeede Hajhoseeny 1, Navid Nasirizadeh* 2, Saeed Rezaei-Zarchi 3,

reason for this new result returns to this fact that structural differences

Mostafa Rezaei-Tavirani 4, Mohammad Atyabi 5, Saber Imani 6

from the hydrogen bond point of view and natural orbital populations affect the proton affinity of the studied amino acids. Atom in molecule

1. Science and Research Branch, Islamic Azad University, Tehran, IR

(AIM) and Natural population analysis (NBO) on all structures show

2. Science and Research Branch, Islamic Azad University, Yazd, IR

that the β structure possess the maximum hydrogen bonds with high

3. Department of Biology, Payame Noor University, Yazd, IR

electron density relative to other second structures which induces a

4. Proteomics Research Center, School of Medicine Shahid Beheshti

more positive charge on the acidic hydrogen of the amino acid.

University, Tehran, IR 5. Department of Biotechnology, Pasteur Institute, Tehran, IR

Keywords: pKa, SCRF, Second Structure, Amino acid, Lysine,

6. Chemical Injuries Research Center, Baqiyatallah University of

Arginine.

Medical Sciences, Tehran, IR (E-mail: [email protected]) The bare surfaces of electrode are not suitable in electrochemical

Abstract No.223

survey of proteins. This, not only leads to decreasing the speed for Effects of some Flavonoids on the Mushroom

irreversible absorption of protein on the surface of the electrode,

Somayeh Hosseini* 1, Nematollah Gheibi 2, 1

Gholamreza Rezaei Behbehani , Majid Sirati Sabet

electron transfer between electrode and protein but also can lead to

3

accompanying

the

conformation

changes

and

loss

of

protein

activity.Hence, it is necessary to provide required groups for the active

A95


communication with macromolecules on the surface of electrode. In

fluorescence and Far-UV CD showed that sucrose increases the PSAO

this project, the structures of nafion nanocomposite polymer and

stability as a concentration dependent manner.

toluidine blue- mediator (which causes catalyzing oxide reaction and restoring protein) are investigated. Then by modifying the surface of

Keywords: Osmolyte, Stabilization, CU-amine Onidase, Thermal

electrode we make it possible the observation of redox enzyme metalo-

unfolding.

proteins reaction through electrochemistry which can be useful in bioelectrochemistry applications.In the first phase of this project, the surface of electrode is modified with nanocomposite polymer nafiontoluidine

blue.

Then

we

examine

the

surface

of

Abstract No.226

obtained

nanocomposite using Cyclic Voltametry, Cronoamperometry, DPV and

Exploring Serpin Inhibitory Mechanism by Molecular Dynamics

electron microscopy data. Our data confirms the formation of

Simulation

nanoparticles of nafion-toluidine blue in the size (dimensions) of 70 nm. We observe that toluidine blue can move the peak of cathode

Zeinab Bagheri 1, Bijan Ranjbar* 1,2, Mohamad Ganjalikhany 1,

enzyme catalas to 100 mv. Then, by stabilizing the enzyme catalase

Tahereh Tohidi Moghadam 3, Zahra Karami 1

we measure the hydrogen proxide (as a substrate of this enzyme) and introduce this biosensor with the detection limit of 0/28 µM.

1. Department of Biophysics, Faculty of Biological Sciences, Tarbiat

Keywords: Nanobiosensor, Hydrogen Peroxide, Catalase Polymer

2. Department of Nanobiotechnology, Faculty of Biological Sciences,

Modares University, Tehran, IR Nafiyn, Toluidine blue.

Tarbiat ModaresUniversity, Tehran, IR (E-mail: [email protected]) Serine protease inhibitor (serpin) is a family of structurally related

Abstract No.225

proteins that control many physiological reactions by inhibition the Purification of Amine Oxidase from Pea Seedlings and Effects

protease. This inhibitor is found in many organisms, from nematode to

of Sucrose on its Stability

human. However, a detailed mechanism of inhibition, how a protein could inhibit a protease, is yet to be understood. In a well known

Aboozar Barzegar* 1, Mojtaba Amani 1, Mohsen Arzanlou 1, Mehdi Zeinoddini

2

mechanism, serpin binds to the active site like a substrate, leading to small conformational change required to lock the protease. On the other hand, hydrogen bond network around the active site of serine

1. Department of Biochemistry, Ardebil University of Medical Science, IR

protease family plays an important role in stabilization of the transition

2. Malekashtar University of Technology, Tehran, IR

state. Herein, to explore mechanism of Serpin’s inhibition, we studied


the effect of binding of ecotin (a natural protein in the periplasmic space of Escherichia coli with a broad range of inhibition) on changing

The copper amine oxidase (AO, E.C 1,4,3,6) is found in all known

of hydrogen bond network near the active site of the thrombin (a key

organisms including planrts, mammals and microorganism. There is an

enzyme in the processes of thrombosis and haemostasis). Three all

increasing interest in amine oxidases due to their involvement in

atomic molecular dynamic simulations of 10 nanosecond were

polyamine metabolism correlated in physiopathological processes.

performed on three forms of thrombin enzyme (free enzyme,

Osmolytes as solvent additives favorably affect protein stability

substrate-enzyme and

extreme environmental stress without any enzymatic activity.Amine

hydrogen bonds were selected around the active site. Distance

oxidase was purified from pea seedlings. Residual activity of pea

changes between the donor and acceptor in the length of trajectory

ecotin-enzyme

complex).

More

than 20

seedling amine oxidase (PSAO) was investigated as a function of

was extracted and distance-distance correlation (DDC) was computed.

various sucrose concentrations and incubation time in different

Affected hydrogen bond by ecotin binding was detected by comparison

temperature. Then for more elucidation of the effect of sucrose the

of the DDC profile amongst the three structures. Results of this

studies were extended using by far-UV CD, fluorescence spectroscopy.

investigation revealed that change in the hydrogen bond network has

PSAO loses its activity by incubation in 63 ˚C for 60 min. Presentation

the primary role in enzyme catalysis.

of sucrose increases the stability of PSAO in terms of activity losing temperature and incubation time. More detailed studies using

A96

Keywords: Molecular Dynamic Simulation, Serpin, Ecotin, Thrombin.


administered with non-steroidal anti inflammatory agents. one of these

Abstract No.227

enzymes is Strain-dependent extracellular metalloproteases which Disulfide Bonds Modulate Dynamics of Catalytic site in Native

secreted by varied S.marcescens strains and has been purified and

State of Pseudomonas Aeruginosa Elastase

characterized by some scholars. This metalloprotease (serrapeptidase) is an important pharmaceutical agent. Serrapeptidase is useful for a

Hossein Rahmani*, Ammar Mohseni, Majid Taghdir, S. Mohsen Ashrafi

wide range of inflammatory conditions. It is an effective drug for the treatment of breast engorgement. serrapeptidase relieve swelling and

Department of Biology, Faculty of Science, University of Guilan, Rasht, IR

pain after surgery buccal, swelling, after maxillary sinus antrostomy.


Enzyme may be effective to break down atherosclerotic plaques and fibrin on the inside of the arteries. In general, this major

Pseudomonas aeruginosa Elastase (PAE) is a Thermolysin like protease

metalloprotease (SMP) has been detected in various strains of S.

(TLP) containing two disulfide bonds. A disulfide bond is located

marcescens which is now manufactured for commercial exploitation

between Cys30-Cys57 which connects two b-strands within the N-

because of its economic importance. In this research, the first,

terminal domain, while another disulfide bond, Cys270-Cys297, is

protease gene encoding a zinc-metalloprotease from the red-

located close to the end of C-terminal domain and connects two a-

pigmented Serratia marcescens ZF03, which is isolated from hot-spring

helices. Previous experimental studies have demonstrated the role of

waters has been sequenced and reported to the GenBank. This

disulfide bonds in enzyme stability and activity. In this study a series of

fragment encodes an extracellular zinc-metalloendopeptidase with a

short time molecular dynamics simulations were performed in the

molecular weight of approximately 50 kDa. This metalloprotease

native and disulfide bond broken states at 298 and 333 °K. Results

purified by ammonium sulphate precipitation, dialysis and DEAE-

revealed that removal of the disulfide bonds in the native state cause a

Sepharose chromatography and characterized. Proteolytic activity was

significant change in dynamics of stability determinant loop. Moreover,

determined by skim milk agar medium and zymography. Protease

flexibility andcorrelation of motions significantly altered within the

activity was optimum at temperature 50-55 ˚C and a range of pH 8-10.

active

The effects of various compounds on protease activity and Kinetic

site

regionin

low

as

well

as

high

temperatures.Theseresultssuggestthatdisulfidebonds are crucial for

parameters

structural stability and activity tuning of PAEin thenative state by

metalloprotease and proteolytic activity, this protease can be used in

adjustingforce distribution in structure.

the pharmaceutical Industries.

Keywords: Disulfide Bonds, Molecular Dynamics Simulation, Stability

Keywords:

Determinant Loop, Correlation of Motions.

protease, Characterization, Pharmaceutical.

Abstract No.228

Abstract No.229

were

determined.

Metallopeptidase,

Considering

Serratia

high

expression

marcescens,

of

Extracellular

Purification and Characterizations of 50-kDa Extracellular

Structural Changes of Ribonucleoprotein ,LMG160, in the

Metalloprotease from Serratia Marcescens ZF03

Presence of Sodium Chloride

Navabeh Salarizadeh 1, Sadegh Hasannia* 2,3, Kambiz Akbari Noghabi 3,

Sayeh Abdossamadi*, Azra Rabbani-Chadegani

Reza Hassan Sajedi 2 Department of Biochemistry, Institute of Biochemistry and Biophysics, 1. Dept. of Biology, Faculty of Science, University of Guilan, Rasht, IR




Modares University, Tehran, IR 3. National Institute of Genetic Engineering and Biotechnology

Low mobility group (LMG) proteins have been studied less intensively

(NIGEB), Tehran, IR

as they are very heterogeneous with low solubility. A fraction of these


proteins named LMG160 (160 kDa) has been isolated from rat liver in pure form and characterized as a ribonucleoprotein (RNP) which

Proteolytic enzymes have been widely used in management of enzyme

detected in RNP-containing nuclear matrix of hepatocyes.In this study,

deficiencies and therapeutic applications. These enzymes are co-

we have investigated the effect of sodium chloride on the structure of

A97


intact and RNase-treated LMG160 using circular dichroism in the range

messenger and apoptosis inducer in cells.Intracellular ROS generated

of far UV (190-260 nm) and fluorescence spectroscopy.The results

by some synthetic benzochromene compounds were measured by a

showed that α-helical content was abundant in both intact and RNase-

non-fluorescence dye, DCFH-DA, which is permeable in cells and

treated LMG160 in the absence of NaCl although the helicity of RNase-

interacts with intracellular ROS to generate fluorescent 2′, 7′-

treated LMG160 was more than intact LMG160. Intrinsic emission

dichlorofluorescein

fluorescence spectra imply that although both emission maxima were

spectrophotometer (λmax = 525 nm). We also measured the nitrite

almost the same, intact LMG160 showed 8 nm red-shift compared to

concentration in the culture medium as an indicator of NO production

the RNase-treated form. It means that accessibility of tryptophan

using the Griess reaction method.Previously we showed that these

residues to the bulk solvent is increased.By increasing sodium chloride

compounds induce apoptosis in these cell lines. In this study we

concentration from 0-1 M, the α-helical content of the intact LMG160

demonstrated that the amount of ROS generations in MCF-7 and MDA-

was decreased almost 8% by increasing %2.1 β-sheets, %1.3 β-turn

MB cells treated for 4 and 24 h were significance and time-dependent.

and %4.7 random coil. The addition of increasing amount of sodium

Nevertheless, T47D cells that were treated for 4 h didn’t produce ROS

chloride to the intact and RNase-treated LMG160 exhibited 50% and

but after 24 h ROS production was considerable. None of the cell lines

25% emission maxima reduction, respectively. Using modified Stern-

produced NO after 4 h. NO generation by MCF-7 cell line treated for 24

Volmer equation, intact and RNase-treated LMG160 Ksv was 6.19M-1

h, was significant. However, these compounds did not induce NO

and 1.67M-1, respectively. Also the amount of fa parameter indicated

generation in MDA-MB231 and T47D cell lines.This study indicated that

that the fraction of the initial fluorescence that is accessible to NaCl is

one of the ways that these compounds can induce apoptosis is by

66% further for the intact LMG160 compared to RNase-treated.The

increasing ROS generation. However this needs to be worked on in

result suggests that RNA moiety is important in this ribonucleoprotein

detail.

(DCF).

DCF

can

then

be

measured

by

structure implying that the intact LMG160 exhibits more relaxed structure compared to RNase treated protein.

Keywords: Breast Cancer, Benzochromene Derivatives, ROS, NO.

Keywords: Ribonucleoprotein, Low Mobility group (LMG) proteins, Circular dichroism, Fluorescence spectroscopy, Ionic strength.

Abstract No.231 Study on the Interaction of L-Proline-Derived Aminophosphinic Acid Ligand with Bovine Serum Albumin

Abstract No.230 The Effect of some Synthetic Benzochromene Compounds on

Fakhrossadat Mohammadi*, Babak Kaboudin, Khavar Moradi,

ROS and NO generation in Breast Cancer Cell Lines (MCF-7,

Mohammad Reza

MDA-MB231 and T47D) Department of Chemistry, Institute for Advanced Studies in Basic

Asma Kheirollahi*, Sussan K.Ardestani

Sciences Zanjan, IR (E-mail: [email protected])

Depatment of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IR (E-mail: [email protected])

Organo-phosphinic acids are

known

as organic derivatives of

hypophosphorus acid (H3PO2) and have found potential applications in the areas of industrial, agricultural and medicinal chemistry. The

The cytotoxic activity of many chemotherapeutic drugs is resulted of

structure of the phosphinic functional group mimics the transition state

their potential in apoptosis induction. Apoptosis is defined as

of peptide hydrolysis. Among the a-functional phosphinic acids, a-

programmed cell death that can occur in response to a variety of

aminophosphinic acids are an interesting class of compounds

insults, such as cytotoxic compounds. Reactive oxygen species (ROS)

possessing broad biological activities. Herein, we report synthesis and

and nitric oxide (NO) generation are some of the signs observed in

the study on the interaction of a carrier protein with a new L-proline

cells subjected to anticancer drugs treatment. In contrast to their role

based aminophosphinic acid ligand. Serum albumins are one type of

on promoting cell growth under non-stress conditions, ROS are

proteins possessing various physiological functions. Serum albumins

powerful inducers of apoptosis when cells are under stress. Nitric

act as carriers for transporting of many of compounds such as fatty

oxide, which is one of the smallest biological products of mammalian

acids, amino acids and drugs. Bovine serum albumin (BSA) is used in

cells, plays various roles such as an intracellular or transcellular

biophysical and biochemical studies as a model protein because of low

A98


cost, ready availability and similarity to human serum albumin (HSA).

of increasing concentrations of 1,3-diaminopropan, 1,4-diaminobutane

In the present study, we investigated the interaction of the synthesized

(putrescine) and 1,5-diaminopentane (cadaverine) in PEM buffer

phosphinic acid with BSA using steady state fluorescence, synchronous

(100mM PIPES, 1mM EGTA, 2 mM MgSO4) using spectrophotometer

fluorescence, and fluorescence resonance energy transfer (FRET). The

UV-vis at 37oC. The results showed that diamines causes a change in

fluorescence titration experiments showed the quenching effect of the

GTP spectrum with a concentration-dependent manner showing

considered synthesized phosphinic acid on the emission of BSA with

interaction of diamines with guanine base of GTP molecule. But quality

slightly blue shift. The binding parameters including number of binding

of the interaction differed from cadaverine to other diamines. In

sites and binding constant have been estimated from the fluorescence

conclusion, diamines interact with GTP molecule probably via

quenching results. Further, the distance of the bound ligand from the

electrostatic interactions with its guanine base. Such interactions may

tryptophan

disturb GTP binding to other molecules.

residues

and

Förster

critical

distance

have

been

determined. The changes in the microenvironment of the tyrosine and tryptophan residues have been investigated using the synchronous

Keywords: Diamines, Putrescine, Cadaverin, GTP, Interaction.

fluorescence spectra. Keywords: Aminophosphinic acid, Bovine Serum Albumin, Ligand

Abstract No.233

binding, Fluorescence Spectroscopy. Polyanionic Couted Nanoparticles Triggers Tau Protein Fibrillization in Vitro Abstract No.232

Mohammad Ali Nasiri Khalili 1, Gholam Hossein Riazi* 1, Shahin Ahmadian 1, Reza khodarahmi 2

A Spectroscopic Study on Interaction of Diamines with Guanosine Triphosphate

1. Institute of Biochemistry and Biophysics, Department of

Tayebe Cheraghi-Shavi 1, Seyede Zahra Moosavi-Nejad* 1,

Biochemistry, University of Tehran, IR

Roya Mahinpur 2, Gholamhosein Riazi 3

2. Faculty of Pharmaceutical Sciences and Medical Biology Research Center (MBRC), Kermanshah University of Medical Sciences,

1. Department of Biology, Faculty of Basic Sciences, Alzahra University,

Kermanshah, IR

Tehran, IR


2. Department of organic chemistry, Faculty of Chemistry, Kashan University, Kashan, IR

Neural transmission is vital process in brain function. Microtubules and

3. Institute of Biochemistry and Biophysics, University of Tehran, IR (E-mail: [email protected])

MAP

proteins

are two

of

the

main macromolecules,

facilate

transmission through neural axons. Among microtubule associated proteins, fibrillar tau protein has been demonstrated to participate in

Diamines are positively charged small molecules that have essential

Alzheimer disease. To mimic tau fibrillization in vivo, several molecules

roles in cell growth, cell division, differentiation, gene regulation,

have been tested for identification of tau aggregation in vitro. In this

enzyme activity and signal transduction. Diamines bind to negatively

study carboxylate couted carbon nanotubes to simulate microtubules

charged

and

and magnetic iron nanoparticles (polyaspartated, polysulfonated and

phospholipid membranes and cause physiological effects in organism.

carboxylated) were employed instead of heparin. The interaction

Diamines are able to make complex, through their amine groups, with

between recombinant human tau and polyanionic nanoparticles were

nucleotide compounds. Positive charge of the amines can interact with

characterized

negative charge of phosphate of nucleotides. Moreover free electron

spectroscopial methodologies. Our results showed that functionalized

pair of nitrogen of the amine group interacts with nucleotide base via

nanoparticles and carbon nanotubes induce tau fibrillization in vitro.

macromolecules

including

proteins,

nucleic

acids

by

using

transmission

electron

microscopy

and

van der Waals interactions. It is expected that diamines affect on nucleotides function either the nucleotides act as a substrate (ATPase,

Keywords:

RNAse) or a ligand (GTP-binding proteins or microtubules). Guanosine

Fibrillization.

Tau

Protein,

Nanoparticles,

Carbon

Nanotube,

triphosphate (GTP) is one of important nucleotides in metabolism.In this research GTP was used as a model nucleotide to investigate GTPdiamine interaction by measuring ∆A253 (lmax of GTP) in the presence

A99


regulates AFP gene expression through binding to the AT motif

Abstract No.234

competitively with hepatocyte nuclear factor 1(HNF1) .ATBF1 protein is Mutation Analysis of Tumor Suppressor Gene PTEN in a

known to interact with various proteins including c-Myb and PIAS3.

Random Population of Iranian Patients with Gastric Cancer

ATBF1 represses c-Myb transcription activity through protein–protein interactions, which may in turn result in the suppression of cell growth

Elham Sadat Hossayni*, Mahmoud Ghaffari, Abed Ali Ziaee,

and differentiation. Moreover, ATBF1 cooperates with p53 to activate

Jamshid Davoodi

the p21Waf1/Cip1 promoter and trigger cell cycle arrest. α-Fetoprotein (AFP) producing gastric cancers are aggressive tumors with venous

Department of Biochemistry, Institute of Biochemistry and Biophysics,

and lymphatic invasion and hepatic metastasis. The goal of the


present study is to investigate whether somatic changes in the AT


motif binding factor-1(ATBF1) gene in exon 9 and 10 and LOH analysis with two microsatellite markers D16S3066 and D16S3139 in the

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is

development or progression of gastric cancer. Until now we have

a tumor suppressor gene that encodes a dual-specificity protein

searched for allelic loss (LOH) with the microsatellite marker D16S3066

phosphatase which contributes to regulation and propagation of signal

at the ATBF1locus by single-strand conformational polymorphism

transduction through the PI3K/AKT signaling pathway.The defects in

(SSCP) and sequencing methods in tumor and blood samples of 14

this gene, for example mutations and deletions, are responsible for the

different patients with gastric cancer. We haven’t found any LOH in

development of some advanced cancers including endometrial cancers,

tumor samples yet. However, we need to study more samples to

ovarian cancers, and glioblastomas.The aim of this study is to

investigate the correlation between progression of gastric cancer and

investigate the significance of PTEN gene mutations on occurrence and

allelic loss in this region in Iranian patients.Methods: DNA extraction

development of gastric cancer.Exon 5 and 7 of PTEN gene were

from blood and tumor tissues, PCR of DNA samples, SSCP of PCR

screened for mutations by " PCR-SSCP-DNA sequencing" followed by

products and sequencing of DNA samples.

silver staining in 94 and 50 patients respectively with pathologically proven gastric carcinoma.No mutations were found in the regions

Keywords: AT motif-binding factor 1(ATBF1), D16S3066, D16S3139,

mentioned. Our initial results suggest that there is no significant

Gastric Cancer.

correlation between mutation in exon 5 and 7 of PTEN gene and occurrence and development of gastric cancer in Iranian patients. But, the exact results need further analysis. Keywords: PTEN, Tumor Suppressor Gene, Gene Mutation, Gastric Cancer.

Abstract No.236 Erlotinib-Protein Binding: HPLC Method Development and Validation

Soheila Bolandnazar* 1,2, Parvin Zakeri- Milani 1, Adeleh Divsalar 2, Hadi Valizadeh 1

Abstract No.235 Studying of ATBF1 Mutations in a Random Population of

1. Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, IR

Iranian Gastric Cancer Patients

2. Dept. of Biological Sciences, Tarbiat Moallem University, Tehran, IR (E-mail: [email protected])

Fatemeh Norouzi, Mahmood Ghaffari*, Abed Ali Ziaeei Lung cancer is the leading cause of cancer-related mortality worldwide, Institue of Biochemistry and Biophysic, Tehran university, IR

for both men and women. Erlotinib a reversible tyrosine kinase


inhibitor, which acts on the epidermal growth factor receptor (EGFR) is almost a new drug used for the treatment of non-small cell lung cancer

AT motif-binding factor 1 (ATBF1) is a transcription factor on 16q22

after the failure of more than one or two courses of previous

region with tumor suppressor activity that contains 4 homeodomains

chemotherapy. The binding of drug to plasma proteins can influence its

and 23 zinc-finger motifs . ATBF1 was identified as a transcription

action and pharmacologic response. Therefore protein binding studies

factor that binds to an AT-rich region, known as the AT motif in the α-

would be of great importance from the clinical point of view. In the

fetoprotein gene promoter region.This transcription factor, down

present study a simple and rapid reversed-phase high performance

A100


liquid chromatographic (HPLC) method with UV detection at 330 nm

carried out to give information on the variations of HAS accessible

was developed for detection of dialyzed Erlotinib in protein-binding

hydrophobic areas and compactness of the protein. The increase in

studies in the presence of albumin. Equilibrium dialysis method with

ANS emission indicated that the drug affects on the hydrophobic

fast spin dialyzer and 25 KD Nitrocellulose filters were used. A

accessible surface area of HAS and leads to exposing of its

reversed-phase Symmetry C18 column (250 mm x 4.6 mm, 5 µm) was

hydrophobic groups.

used at room temperature. The mobile phase was a mixture of methanol, acetonitril and potassium dihydrogen. Analysis was run at a

Keywords: Erlotinib, HSA, Interaction, Spectroscopy.

flow rate of 1.3 ml/min. The run time for Erlotinib was approximately 7 minutes. The method was validated for its specificity, linearity, accuracy and precision.Therefore a simple, accurate and precise

Abstract No.238

reversed-phase isocratic HPLC method with UV detection has been optimized and validated for the determination of erlotinib in human

Rolling Circle Amplification Technique in Medical Diagnosis

plasma.

Vahid Hamidi*, Hedayatollah Ghourchian Keywords: Erlotinib, Protein-binding, HPLC, Plasma. PTEN, Tumor Suppressor Gene, Gene Mutation, Gastric Cancer, IR (E-mail: [email protected]) Abstract No.237 Due to its robustness and simplicity, the Rolling-Circle Amplification Interaction of Erlotinib Hydrochloride with Human Serum

(RCA) of circular DNA probes holds a distinct position in DNA based

Albumin

diagnostics among other isothermal detection methods. RCA reactions exhibit an excellent sequence specificity that is favorable for

Arash Khodaei*, Leila Hassani, Akram Hamidi,

genotyping or mutation detection, antigen detection, DNA based

Elham Safar gholizadeh, Sevda Yousefian

biosensors and allows to unambiguously identification of DNA markers on the excessive unrelated background. We use circular DNA as a

Department of Biological sciences, Institute for Advanced Studies in

template for RCA reaction. When primers anneal to the template in

Basic Sciences (IASBS), Zanjan, IR

presence of nucleotides, reaction buffer and phi29 DNA polymerase,


RCA proceeds and make long strand DNA. In the presence of two primers, a complex pattern of DNA strand displacement ensues that

Erlotinib hydrochloride is a drug used to treat non-small cell lung

generates 109 or more copies of each circle in 90 minutes. Using a

cancer (NSCLC), pancreatic cancer and several other types of cancer.

single primer, RCA generates hundreds of tandem linked copies of a

Erlotinib specifically targets the epidermal growth factor receptor

covalently closed circle in a few minutes. An important factor for the

(EGFR) tyrosine kinase, which is highly expressed and occasionally

success of this method is the unique nature of phi29 DNA polymerase

mutated in various forms of cancer. The human serum albumin (HSA)

which has excellent strand displacement activity. The use of primers

is a major plasma protein. It is named a multifunctional plasma carrier

with 3´ thiophosphate-protected ends is also important, allowing

protein because of its ability to bind to an unusually broad spectrum of

circular DNA molecules to be amplified at least 10,000-fold by

ligands.

their

protecting the primers from the 3´_exonuclease activity of phi29 DNA

erlotinib

polymerase. To achieve amplification, phi29 DNA polymerase appears

hydrochloride as a drug with human serum albumin is significant. In

to initiate multiple replication forks on each circle and to perform

this study, experimental investigation on the interaction of erlotinib

exponentially cascading strand displacement amplification. These

hydrochloride with human serum albumin was carried out.Stern–

results indicate that circular DNA probes can be amplified to the high

Volmer dynamic quenching constant, binding constant and the number

levels required for solution based DNA diagnostics.

HSA

binds

pharmacokinetics.

to

Thus,

the the

number

of

drugs

interaction

altering

between

of binding sites for interaction of erlotinib hydrochloride with HSA were measured using analyzing of the fluorescence spectroscopic data. The

Keywords: Rolling Circle Amplification, Phi29 DNA polymerase,

intrinsic

Displacement Activity, Isothermal Amplification.

fluorescence

spectra

indicated

that

the

intensity

of

fluorescence emission decreases as a function of erotinib concentration indicating the partial opening of the protein structure upon interaction with the drug. Fluorescence measurements on HSA–ANS complex was

A101


Abstract No.239

Lysozyme,

Keywords:

Curcumin,

Fluorescence

Spectroscopy,

Molecular Docking. Analysis of the Binding Interaction of Curcumin with Lysozyme Abstract No.240

Afshin Mahmoudian*, Fakhrossadat Mohammadi Structural and Functional Study of Lysozyme from Rutilus Department of Chemistry, Institute for Advanced Studies in Basic

Frisii Kutum

Sciences (IASBS), Gava Zang, Zanjan, IR (E-mail: [email protected]) Curcumin

Faezeh Moradi*, Mahmoud Reza Aghamaali, Reyhaneh Sariri

[1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-

Department of Biology, Faculty of Science, University of Guilan, Rasht, IR

dione] is a polyphenol compound extracted from the herb Curcuma


longa Linn. Modern scientific community discovers that curcumin exhibits an enormous variety of pharmacological and biological

Lysozyme is an antibacterial protein, has been implicated in innate

activities including anti-oxidant, anti-tumor, anti-inflammatory, anti-

immunity in invertebrates, but its activity in shrimp and some other

bacterial, and anti-protozoal activity. Further, curcumin has the

marine animals, remained to be determined. We are going to clone the

possibility to slow down the progress of Alzheimer's diseases by

white Rutiluslysozyme cDNA using a PCR strategy following to

reducing b-amyloid formation. Lysozyme is an antimicrobial proteinase

designing sutable primers according to the nearest spesiec to Rutilus,

that has ability to lyse the cell walls of bacteria by hydrolyzing the

and detected its activity in haemocytes using a lytic-zone assay against

bond between N-acetylglucasamine and N-acetylmuramic acid of the

Micrococcus luteus, The deduced amino acid sequence resulted in 150

peptidoglycan. Lysozyme consists of a single chain polypeptide

amino acid with 46% identity to hen egg white lysozyme. RT-PCR was

containing 129 amino acid residues which is crosslinked with 4 disulfide

used to detect lysozyme mRNA in haemocytes. Analysis of the amino

bridges. The investigation of interactions between small molecules and

acid sequence of the lysozyme may be showed that it belongs to the

lysozyme has an important meaning on realizing the transport and

C-type family of lysozymes. Furthermore, the lysozyme amino acid

metabolism process of the small molecules and the relation of

sequence contained extra residues at its C-terminus, which are

structure and function of lysozyme. With respect to the different

characteristic of marine invertebrates. This information will be useful in

physiological and pharmaceutical functions of lysozyme and its widely

future studies on the molecular mechanisms of immunity in marine

distribution in various biological fluids and tissues including avian egg,

invertebrates.

animal secretions, human milk, and tears, we decided to study the interaction of curcumin with chicken egg white lysozyme. This study

Keywords: Lysozyme, Shrimp, Prawn, Purification, Haemocyte, PCR-

was carried out using different techniques including steady sate

cloning, EST.

fluorescence, fluorescence,

synchronous UV-vis absorption,

fluorescence,

three-dimensional

fluorescence resonance energy

transfer, and molecular docking. The fluorescence experiments

Abstract No.241

revealed that addition of curcumin effectively quenched the intrinsic fluorescence of lysozyme by formation of a non-fluorescent complex

The Effect of di- and Polyamines on 2΄,3΄-cyclic Cytidine

(static quenching). The number of substantive binding sites and the

Monophosphate: a Spectroscopic Study

binding constant were calculated by relevant fluorescence quenching data. Based on the Förster’s theory of non-radiative energy transfer,

Mona Amuri, Seyede Zahra Moosavi-Nejad*

distance between the donor (lysozyme) and acceptor (curcumin) as well as the critical energy transfer distance has also been calculated.

Department of Biology, Faculty of basic Sciences, Alzahra University,

The molecular docking studies revealed that specific interactions were

Tehran, IR

observed with the Trp-62 and Trp-63 residues. The calculated


thermodynamic parameters showed that H-bonds and van der Waals interactions played a major role in stabilizing the curcumin–lysozyme

The nucleotide 2΄,3΄-cyclic cytidine monophosphate is produced

complex.

during hydrolysis process of RNA by RNAse A, although this compound is an intermediate and hydrolyzed in the next step. A large number of

A102


studies (e.g. effect of different ligands and their interactions) have

structures of proteins. A Furin cleavable linker is used for evaluation of

been carried out on it. On the other hand, polyamines are produced

structures. Best scored models are studied for their stability and

naturally in living cells. They are implicated in a wide range of cell

original structures by over fitting of models and initial structures,

processes including cell growth, cell division, differentiation, gene

solvent accessible surface and ramachandran plot. Results demonstrate

regulation, enzyme activity and signal transduction.In this study effect

that presence of StxB at the N-Terminal of structure stabilizes the total

of various concentrations of di and poly-amines has been investigated

fusion because of C-Terminal structure of StxB. Number of linker

on the structure of 2΄,3΄-cyclic cytidine monophosphate by following

repeats must be at least one but more than 3 repeats those not

∆A284. 2΄,3΄-cyclic cytidine monophosphate and all amines were

change the structure stability.

dissolved in Tris-EDTA buffer (Tris 100mM, EDTA 2mM, pH 7.5). As our results showed, none of the diamines, including 1,3-diaminopropan,

Keywords: CtxB, StxB, Modeling, Furin linker, Shigella, Cholera.

1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine), had significant effect on the nucleotide spectrum, while both of polyamines

(spermidine

and

spermine)

caused

significant

and

Abstract No.243

meaningful change on 2΄,3΄-cCMP absorbance. Overall, it seems that polyamines interaction with phosphate group and/or cytosine base of

Spectroscopic Investigation on the Interaction of

the nucleotide resulting in a change in its absorbance while diamines

c-MYC quadruplex DNA with Water-Soluble

may interact only with phosphate group of the nucleotide which has no

Tetrapyridinoporphyrazinatozinc(II)

effect on its absorbance. This difference may be related to size of polyamines that are larger than the diamines.

Zahra Fazeli* 1, Laila Hasani 1, Elham Safaei 1, Hossein Rastegar 2, Minoo Akbari 2

Keywords:

2΄,3΄-cyclic

Cytidine

Monophosphate,

Diamine,

Polyamine, Interaction.

1. Department of Biological sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, IR 2. Food and Drug Control Research center, FDCL MOH, Ministry of health and Medical Education, Tehran, IR

Abstract No.242

(E-mail: [email protected]) In Silico Analysis of Conformational and Immunological Differences of Linked StxB and CtxB

Nucleic acid sequences which are rich in guanine are capable of forming four-stranded structures called G-quadruplexes. Oncogenes

Hojat Borna 1, Hosein Honari* 2, Seied Jafar Mousavy 2

are especialy rich in quadruplex. It is known that ~90% of c-MYC transcription

1. Cellular and Molecular Biology, Imam Houssein University, Tehran, IR

composed

is controlled of

by a

consecutive

five

27-nt

purine

guanine

rich

strand

stretches.

is

Recent

2. Faculty Member of Biology Group and Research Center, Imam

experimental data suggest that even a brief inhibition of c-MYC

Houssein University, Tehran, IR

expression may be sufficient to permanently stop tumor growth and


induce regression of tumors. The ligands that bind to hypersensitivity element III1 (NHE III1) of the c-MYC promoter can control the

CtxB and StxB are of the most impacting factors in initiation of cholera

transcriptional activity of the c-MYC oncogene. Here, interaction

and shigella infections. These two proteins are working as carriers of

between

catalytic domains of shigella and cholera toxins. All such proteins have

pyridinoporphyrazinatozinc(II) {[Zn(3,4-tmtppa)]4+} and c- MYC G-rich

5 same subunits along with a catalytic domain among. These two

oligonucleotide was investigated. The absorption spectrum of {[Zn(3,4-

proteins are considered as novel targets for immunological studies. It

tmtppa)]4+ displays a Q band at ~670 nm. It was found that at low

is assumed that accompaniment of both of proteins can boost the

concentrations of DNA, a hypochromicity in the Q band of the complex

immunological effects. Linking both proteins with a linker is a possible

is shown but, at higher concentrations, the intensity of spectra

solution, but structural integrity and proper folding of proteins must

increases and the maximum absorption shifts to higher wavelengths

remain stable. In order to discover suitable number of linker molecules

considerably. It seems two types of complexes form due to interaction

for proper separation of proteins, CtxB-linker(n)-StxB and StxB-

of the porphyrazine with c-MYC G-quadruplex DNA. The quenching of

Linker(n)-CtxB manner of proteins are designed and structures

[Zn(3,4-tmtppa)]4+ by G4 DNA and the G4–thiazole orange by the

modeled with Modeller 9 program based on protein data bank

complex were measured by fluoresnce spectroscopy. Stern–Volmer

water-soluble

N,N′,N″,N′″-tetramethyltetra-3,4-

A103


dynamic quenching constant, binding constant and number of binding

converted with time into a highly ordered β-sheet. The Combined effects

sites for the interaction of {[Zn(3,4-tmtppa)]4+ with the G-rich

of hydrophobic and electrostatic interactions, both of which are

oligonucleotide, its complementary C-rich strand and

the DNA

strengthened by presence of the alcohols, may drive the protein toward

structure formed by mixture of G-rich and C-rich oligonucleotides were

amyloid formation. Subtle balance between these two types of

measured using analyzing of the fluorescence spectroscopic data.

interactions may determine whether the fibrils or amorphous aggregates dominate as end products.

c-MYC,

Keywords:

Quadruplex,

Interaction,

Spectroscopy,

Porphyrazine.

Keywords: Amyloid, Apo-Carbonic Anhydrase, Alcohols, Hydrophobic Intraction.

Abstract No.244 Abstract No.245 Amyloid-like Aggregate Formation in Apo-Carbonic Anhydrase Using Different Alcohols

Effect of Glycine on Human Serum Albumin Glycation

Ali Eshaghi* 1, Marjan Sabbaghiyan 2, Azadeh Ebrahim-Habibi 3,

Asghar Farajzade*, S. Zahra Bathaie

Mohsen Nemat-Gorgani

4

Department of Clinical Biochemistry, faculty of Medical Sciences,

1. Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, IR

Tarbiat Modares University, Tehran, IR (E-mail: [email protected])

2. Department of Andrology, Reproductive Biomedicine Research

Diabetes is one of the prevalent diseases in a lot of countries and makes

Center, Royan Institute, ACECR, Tehran, IR

huge expenses to human beings. High blood sugar plays a key role in

3. Endocrinology and Metabolism Research Center, Tehran University

diabetes’s complications because sugars with the reducing end interact

of Medical Sciences, Tehran, IR

with biological macromolecules, e.g. proteins and produce advanced

4. Stanford Genome Technology Center, Stanford University, Palo Alto,

glycation end products (AGEs). In addition, AGEs and their receptors are

CA, USA, US

involved in the pathogenesis of heart failure, cancer, Alzheimer’s disease,


Parkinson’s disease, familial amyloid, polyneuropathy, prion disease, and etc. It has been clear that inhibiting or decreasing the AGEs production

Amyloid fibrillation plays a crucial role in disorders such as Alzheimer’s

helps to decrease the diabetic complications like cataract, nephropathy,

and Parkinson’s diseases. However, despite the ability of most proteins to

retinopathy, atherosclerosis, and etc. We have shown before that

form amyloid fibrils, not much is known about their structures and

chemical chaperones e.g. glycerol and spermidine are able to reduce

factors that contibute to their formation. Organic solvents, including

hemoglobin glycation by glucose (Glc) and glucose-6-phosphate (Glc-6-

alcohols could be used to induce fibrillation in proteins. In this study the

ph). In continue, the effect of other chemical chaperons on glycation of

effects of various alcohols were compared on apo- carbonic anhydrase

proteins are studding in our lab. The results of glycine (Gly) application,

amyloid formation. Congo red and thioflavin-T binding and CD

as a member of this family of compounds, are presented here. We

spectroscopy experiments suggested that the aggregates induced by

investigated its inhibitory effect on albumin (Alb) glycation by both Glc

alcohols have amyloid-like properties, and atomic force microscopy data

and Glc-6-ph. Therefore, the same concentration of Alb solution was put

indicated

morphology.

in different vials and incubated with Glc / Glc-6-ph, in the presence or

Comparison of stability of aggregate species was also performed. Among

absence of Gly up to four months. Then all samples were investigated by

the different alcohols tested particularly fluorinated alcohols (HFIP, TFE)

fluorometry, CD and electrophoresis. The results showed the various

were particulary effective in amyloid like aggregate formation. At low pH,

degrees of protein glycation by each of the used sugars, also Gly showed

formation of aggregates was promoted by HFIP and TFE with an

different degrees of inhibition of Alb glycation induced by each reducing

optimum at 5% (v/v) and 12% (v/v) respectively. The effective

sugars. In conclusion, Gly as a glycation inhibitor decreased AGE

concentration to drive the protein toward amyloid-like stucture formation

production and conserved protein structure.

that

these

aggregates have a

spherical

was higher for other alcohols.Stability of oligomers that were formed in fluorinated alcohols was significantly greater than those formed in other alcohols. Our results also demonstrate that when the alcohols are added an α-helix is formed at first. The partly α- helical conformation is

A104

Keywords: Glycine, Glycation, Albumin, AGEs, Chemical Chaperon.


In the present work, a halo-alkali tolerant mannan-degrading

Abstract No.246

bacterium (strain FH-1) was isolated from soil samples of Jahrom city, Positive Homotropic Effect in two-phasic Non-Michaelis

Fars, Iran. Phenotypic classification and 16S rDNA sequence analysis

Kinetics of Xanthan Lyase

placed FH-1 in the genus Gracilibacillus. Strain FH-1 grew well at salinities and alkalinities of 0-20% NaCl and pH of 7-10. When grown

Saeedeh Jafari Nodooshan*, S.Zahra Moosavi-Nejad,

on 1% locust bean gum (LBG) as the carbon source at 5 % NaCl and

Mohammad Reza Soudi

pH 9.0, maximal β-mannanase activity was produced in the culture supernatant after 3 days. Partial purification of the enzyme was done

Dept. of Biology, Faculty of Basic Sciences, Alzahra University, Tehran, IR

by a combination of ammonium sulfate precipitation and DEAE-


Cellulose ion exchange chromatography. The endo-1,4-ß-D-mannanase activity of the enzyme was confirmed by using specific substrate, Azo-

Xanthan gum, a bacterial anionic heteropolysaccharide, structurally

carob galactomannan. The optimum temperature and pH for β-

consists of a main chain of D-glucose units which linked by β-1,4

mannanase activity on LBG as a substrate were 50 oC and 10.0

bounds, as it is in cellulose, plus side chains attached to glucose

respectively. The enzyme exhibited its optimal activity at 0-1 M NaCl

residues alternately. Trisaccharide side chain contains of a D-

and 50 % of its initial activity remained when assayed in 2 M NaCl,

glucuronic acid unit between two D-mannose residues. Xanthan,

indicating a high degree of halostability. These results suggest that the

produced

found

β-mannanase secreted by the newly isolated Gracilibacillus sp. strain

commercial applications as a viscosity enhancing agent in aqueous

FH-1 is a suitable candidate for using as a detergent ingredient due to

solutions. Viscosity of xanthan solutions may be controlled via

its activity at a broad pH and NaCl concentrations.

by

xanthomonas

campestris,has

degrading its backbone or side chain by using hydrolyzing enzymes including xanthanase (for hydrolyzing the backbone) or xanthan lyase

Keywords: β-mannanase, Alkalophile, Gracilibacillus, Locust bean

(for hydrolyzing the side chain).In this study, xanthan lyase activity

gum, Halostability.

was measured by monitoring ∆A235 of a solution containing appropriate concentration of xanthan in 50mM sodium phosphate buffer pH=7 and xanthan lyase enzyme secreted by a xanthan lyase

Abstract No.248

producing bacterium at 30 oC. According to our results, the enzyme saturation curve showed non-Michaelis behavior (positive homotropic effect).

More

analysis,

using

Eadie-Hofstee

and

Hill

plots,

Monitoring Surface Plasmon Resonance of Gold Nanorods in Biological Buffers

demonstrated the enzyme shows two phases during saturation by xanthan as substrate, resulting in enzyme activation. We concluded

Hossein Barkheh 1, Bijan Ranjbar*

1,2

, Tahereh Tohidi Moghadam

2

that xanthan is not only the enzyme substrate but also its activator. so that increasing concentrations of xanthan can induce a more active conformation in xanthan lyase.

1. Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR 2. Department of Nanobiotechnology, Faculty of Biological Sciences,

Keywords: Xanthan lyase, Homotropic effect, Non-Michaelis-Menten, Xanthan.

Tarbiat Modares University, Tehran, IR (E-mail: [email protected]) Nobel metal nanoparticles with their shape and size dependent properties have the potential to be exploited in many research areas,

Abstract No.247

such as nanoscale electronics, catalysis, optical sensing, imaging, Isolation of a β-mannanase Producing Bacterium, Secretion

gene/drug carrier, therapeutics etc. In the light of this, nanostructures

Optimization and Biochemical Properties of the Enzyme

of gold with rod morphology (GNRs) have attracted much more attention in novel medical and paramedical applications. It is important

Fatemeh Honari*, Hamid Reza Karbalaei-Heidari

to investigate the stability of GNRs in different biological buffers, since presence of some ions might affect the structure and morphology of

Department of Biology, Faculty of Sciences, Shiraz University, Shiraz, IR

the nanostructures. Herein, we present the effect of three biological


buffers on the longitudinal plasmon resonance of GNRs, to check the sensitivity of LSPR and maintenance of rod morphology. Short gold

A105


nanorods were synthesized according to the conventional seed-

photoprotein. Our finding revealed that the replacement of ASP with

mediated growth protocol and characterized by UV-Vis spectroscopy,

GLY alters dynamics and energetic properties in functional regions of

transmission electron microscopy (TEM), and atomic absorption

the structure and affects emission light and Ca2+- sensitivity

spectroscopy (AAS) for shape, size and yield determination. The typical

properties in photoprotein.

surface plasmon absorption bands in the visible and near IR region confirmed the rod morphology of the nanostructures. Phosphate, Tris

Keywords: Aequorin, Molecular Dynamic Simulation, Ca2+-affinity,

and HEPES buffer solutions were prepared and the pH was adjusted to

Life-time.

be 7.4. Concentration of the buffer solutions was 0.05 M in the final working solutions. Prior to use, GNRs were purified by two rounds of centrifugation at 12000 rpm, for 6 minutes. Pellet of the second round

Abstract No.250

with a fixed concentration of gold nanorods were diluted with each buffer solution. UV-Vis spectra of the interacted samples were recorded

Surface Modification of Gold Nanorods with

to monitor the plasmonic bands. Results showed that neither the

Polyethyleneglycol for Nano Biosensing Applications

transverse SPR nor the longitudinal one have changed notably. Although the intensity of LSPR peak has been somewhat affected in

Tahereh Tohidi Moghadam, Bijan Ranjbar*

buffer medium, the rod morphology is still maintained. Sensitivity of gold nanorods to trace changes in the local environment/ refractive

Department of Nanobiotechnology Faculty of Biological Sciences

index encourages the possibility of utilizing these nanostructures in

Tarbiat Modares University, IR

development of nanostructures for various biosensing applications. Gold

Keywords:

Nanorods,

Surface

Plasmon

Resonance,

Nanobiosensor.

(E-mail: [email protected]) There has been great interest in the domain of nanobiotechnology to design and develop new generation of nanobiosensors. Amongst anisotropic nanoparticles, gold nanostructures of rod morphology (GNRs) have attracted significant attention, for having variety of applications in biomedicine and biosensing. Although many fruitful

Abstract No.249

features of gold nanorods have been introduced so far, the Investigation of Mechanistic Influence of Mutation D117G in

nanostructure itself is believed to show strong cytotoxicity, since it has

Aequorin from Aequorea Victoria: a Molecular Dynamics

been synthesized in the presence of hexadecyltrimethylammonium

Simulation Study

bromide (CTAB). The cationic surfactant is also known to play important role in the stabilization of GNRs, and maintenance of rod

Somayeh Asfiaei*, Ammar Mohseni, Majid Taghdir

morphology.

Herein,

polyethyleneglycol

has

been

utilized

for

neutralization of the positively charged GNRs. Gold nanorods were Department of Biology, Faculty of Science, University of Guilan, Rasht, IR

synthesized according to the conventional seed mediated protocol.


Formation of the rod morphology, size, monodispersity, concentration and yield of synthesis were characterized by UV-Vis spectroscopy,

The photoprotein aequorin, isolated from the jellyfish Aequorea

transmission electron microscopy (TEM) and atomic absorbance

victoria,is

protein

spectroscopy (AAS). Excess CTAB was removed by one round of

apoaequorin and the prosthetic factor coelenterazine that emits light

centrifugation at 12000 rpm for 6 minutes. Sample of GNRs with 75 nM

upon calcium binding. In order to understand the mechanism of the

concentration was interacted with 400 µL PEG-4000 (600 mg. mL-1).

reaction, the study of structure-function relationships was undertaken

The mixture was incubated at ambient temperature for 1 hour. Excess

with respect to modifying certain of its amino acid residues. One of

PEG was removed by another round of centrifugation. Surface plasmon

these mutations is the D117G that is located out of substrate binding

resonance of bare and pegylated GNRs was monitored via UV-Vis

cavity and Ca2+-binding loops. In this study short time molecular

spectrophotometer.

dynamics simulations were performed to investigate the influence of

resonance (TSPR)

this fine change on dynamic properties of substrate binding cavity and

appeared at 528 nm and 708 nm, respectively. Intensity of SPR bands

Ca2+ binding loops that direct emission light properties in this

of the pegylated GNRs decreased upon treatment, without any shift in

photoprotein. Previous studies showed that this mutation decreases

both regions. Although a considerable quantity of the cationic

affinity of loops to Ca2+ and increase half-life of emission light in the

surfactant has been replaced by polyethyleneglycol, stability of GNRs

A106

a

bioluminescent

complex formed with

the

Results

showed

that

transverse

plasmon

and longitudinal plasmon resonance (LSPR)


and their typical SPR absorption bands has not undergone undesirable

Abstract No.252

change. Pegylated gold nanorods could be utilized for in-vivo applications, such as drug delivery with higher circulation time,

Effect of DMSO & Triton on G-quadruplex-Hemin Interaction

photothermal therapy, and nanobiosensors of more specificity in the

Zahra Karami 1, Bijan Ranjbar* 1,

upcoming research fields of nanobiotechnology.

Arastoo Badoei-Dalfard 2 Keywords:

Nanobiosensor,

Gold

Nanorods,

Surface

plasmon

Resonance.

1. Faculty of Biological Sciences, Department of Biophysics, Tarbiat Modares University, Tehran, IR 2. Faculty of Science, Department of Biology,Shahid Bahonar University

Abstract No.251

of Kerman, Kerman, IR (E-mail: [email protected])

Lipase Applying in Soybean Oil to Biodiesel Production and Comparison with Chemical Method

Ahmad Panahazari* 1, Shiva Irani 1, Saleh Soleimani 1, Seyed Mohammad Atyabi 2, Dariush Norouzian 2 1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, IR 2. Pilot Biotechnology Department, Pasteur Institute of Iran, Tehran, IR (E-mail: [email protected])

It has been reported that complexes formed by hemin and some Gquadruplexes can be developed as a new class of DNAzyme with peroxidase activity. DNA in single-stranded form has the ability to fold into complex structures that serve as highly specific catalysts. Here, we report metal ions induced guanine quadruplex formation with d(GTG3TAG3CG3T2G2),

which

shows

peroxidase

function

upon

complexation with hemin.DNA oligomers were heated at 88°C in distilled water and gradually cooled. The DNAs were then treated in HEPES buffer in the presence and absence of DMSO and Triton and

Nowadays, biodiesel is well accepted as a renewable energy. Biodiesel is a fuel comprised of monoalkyl esters traditionally derived from vegetable oils or animal fats. There is currently an unprecedented increased interest , demand for biodiesel and other fuels derived from renewable biomass .Our study was conducted to investigate the optimum conditions for biodiesel formation from soybean oil with chemical reaction and lipase enzyme. The results indicated that increasing of temperature is important factor in both methods. In this study through experimental investigation of reaction conditions such as, reaction temperature which are deemed to have main impact on reaction conversion efficiency. The oil conversion was influenced by the methanol/oil molar ratio. The technical tools and processes for monitoring the transesterification reactions like GC have also been summarized. The experimental results showed that in chemical method a 1:8 molar ratio of methanol and ethanol to oil, addition of 1% wt KOH catalyst, 60 ºC reaction temperature gave the best results, and the biodiesel yield exceeded 95% at 90 min. In enzymatic a 1:4 molar ratio of methanol to oil, 45 ºC reaction temperature are

both of them were kept at room temperature to allow proper folding. An equivalent volume of hemin was added to the G-quadruplex solution, and incubated to form DNAzyme complex.Spectroscopic measurements were carried out in order to characterize complex formation.

The

UV-vis

spectroscopy

results

showed

that

the

uncomplexed hemin has a soret absorption band centered at 397 nm. Upon incubation with G-DNA, in the presence of DMSO and Triton, the absorption center showed a slight red shift to 404 nm with hyperchromisity. This characteristic has been used to investigate the hemin-DNA interaction. However, in the absence of Triton and DMSO, only hyperchromicity and considerable blue shift was observed. By using Triton and DMSO alone, the significant results were obtained. There was no change in the intensity of Hemin-G-quadruplex interaction when using DMSO and hyperchromacity was not observed. But after using Trion in the buffer composition hyperchromacity and red shift was observed in comparison with DMSO. Keywords: Hemin, Deoxyribozyme, Peroxidase, DMSO, DNAzyme.

best condition in biodiesel production. Enzyme stability and activity was investigated in present of methanol and tert-butanol. Methanol play a reducing role in affecting enzyme stability but tert-butanol increased the enzyme activity. Keywords:

Biodiesel,

Triglyceride,

Gas

Chromatography,

Transesterification, Base Methods.

A107


1. Department of Biology, Faculty of Science, The University of Guilan,

Abstract No.253

Rasht, IR Molecular Dynamics Simulation Study of Lipase B from

2. Department of Biochemistry, Faculty of Science, Tarbiat Modares

Candida Antarctica

University, Tehran, IR (E-mail: [email protected])

Mohamad Reza Ganjalikhany, Bijan Ranjbar*, Zeinab Bagheri Mnemiopsin from Mnemiopsis leidy emits light in the presence of Ca2+ Department of Biophysics, Faculty of Biological Sciences, Tarbiat

decomposing into apomnemiopsin, coelenteramide and CO2. To


understand mechanism of the reaction, a comparative structure-


function study was undertaken with respect to two single mutants, Leu36His and Phe186His located in the substrate binding cavity. Our

Candida antarctica lipase B (CalB) belongs to the α/β hydrolase family

experimental results showed that these mutations stop bioluminescent

which is act on the ester bonds of tri-acyl glycerol. CalB is a

reaction. Molecular dynamic simulation studies indicated an increase in

psychrophilic lipase that is able to perform lypolytic activity at low

overall flexibility of Ca2+-binding loops and substrate binding cavity

temperatures. Cold-active lipases have several applications in industry,

residues in mutants compared to the wild type. Further analyses on

production of pharmaceuticals, food, and fine chemical. To understand

Phe186His mutant revealed that mobility of coelenterazine in some

the mechanisms involving in the psychrophily of CalB, molecular

regions and substrate-protein interaction energy is decreased, while

dynamics (MD) simulation has been undertaken to explain its dynamics

these structural properties are not changed in Leu36His mutant. Finally

at atomic level. Crystal structure of CalB was obtained from Protein

these findings revealed that these two substitutions prevent disruption

Data Bank (1TCA) as the initial structure for MD simulations. MD

of peroxide group of coelentrazine and consequently bioluminescent

simulations were performed by AMBER 10.0 with all-atom force filed

reaction by altering the dynamic and energetic properties in this

ff99SB. The structure was hydrated in a 10 Å layer of TIP3P water

region.

model. In this study, several MD simulations have been performed for 30 ns at three different temperatures (5, 35 and 60 °C). A highly

Keywords:

flexible alpha helix (α5 helix) was identified in which act as a lid for the

Study.

Mnemiopsin,

Structure-function,

Molecular Dynamics

active site clef of CalB. According to the results obtained from RMSF graphs, the extent of flexibility is much higher at low temperature rather than higher temperatures. Further investigation of the active

Abstract No.255

site revealed that the starting open conformation of the enzyme become close immediately at 35 and 60 °C while it remains open for a

Artificial Superoxide Dismutase Activity of Copper-Cysteine to

considerable time. Radial distribution function of water molecule in the

Electrochemical Detection of Superoxide

activeite also verifies the movement of the lid. The presence of water molecules at open conformation is higher than that of closed state. It

Fariba Dashtestani 1, Hedayatollah Ghourchian*1,

is suggested that the cold-activity of Calb is tightly related to the

Hossain Ali Rafiee-Pour 2, Khadijeh Eskandari 1

movement of the lid at low temperatures. As it observed in the current study, open state is more stable at 5 °C rather that 35 and 60 °C.

1. Laboratory of Microanalysis, Institute of Biochemistry and, Biophysics, University of Tehran, Tehran, IR

Keywords: Candida Antarctica Lipase B, Psychrophilic Enzymes, Molecular Dynamics Simulation, Open-Closed Conformations.

2. Department of Biotechnology, Faculty of Science, University of Kashan, Kashan, IR (E-mail: [email protected])

Abstract No.254

Superoxide dismutase (SOD) represents an essential defense system against oxygen-derived free radicals, specifically superoxide radical

Molecular Dynamics Study of two Mechanistic Mutations in

anion. Superoxide can initiate a series of free radical reactions, which

Mnemiopsin from Mnemiopsis leidy

causes a vast number of diseases. So, quantitative analysis of in vivo is very important. In most of the analysis methods, SOD was immobilized

A108

Ammar Mohseni 1, Maryam Molakarimi 1, Majid Taghdir* 1,

through cysteine self assembled monolayer on gold (Cys/Au) electrode.

Reza H. Sajedi 2, Saman Hosseinkhani 2

Here, we design and compare three approaches of superoxide


detection by using SOD/Cys/Au, Cu2+/Cys/Au and Cys/Au electrodes.

large and the small subunits of executioner caspases was studied. It

The Cu/Cys/Au electrode shows quasi reversible peaks with formal

was observed that the Smac protein by far is the most potent agent in

potential of 29 mV versus Ag/AgCl at scan rate 50 mVs-1 as same as

reversing caspase inhibition. In addition, Caspase-3 inhibition by XIAP

SOD/Cys/Au electrode. The ampromrtic response for was monitored at

domains was more sensitive to SMAC peptides than that of caspase-7.

an electrode potential 250 mV at pH 7.4 phosphate buffer and 500

Finally, while, BIR1-2 inhibited caspase-3 was very sensitive to SMAC

rpm. In addition, the linear detection range and detection limit of

interference,

superoxide anion radical at Cu2+/Cys/Au electrode were 3.4-254.2 and

antagonism very weakly. These results indicate that under conditions

BIR1-2

inhibited

caspase-7

responded

to

SMAC

2.3 µM respectively. Comparison between voltammograms of different

of extensive XIAP cleavage and involvement of caspase-7 as the

electrodes revealed that current intensity was increasing by the order

driving force for execution of apoptosis, Smac, and by extension Smac

of Cu2+/Cys/Au > SOD/Cys/Au > Cys/Au electrodes. This increasing

based anticancer agents, cannot be effective in inducing cell death.

order was also seen for the amprometric response. The experimental results revealed that Cu2+, either as coordinated with Cys or as SOD

Keywords: Apoptosis, XIAP, Executioner Caspases, Smac Peptides

redox center, plays a critical role in electrochemical response on the

and Protein.

Cys/Au electrode. It seems that in Cu2+/Cys/Au electrode, Cu2+ coordinate with amine and carboxyl groups of Cys and form a complex. Thus, Cu2+/Cys/Au electrode shows better superoxide dismutase

Abstract No.257

activity than SOD/Cys/Au electrode, since Cu2+ in the metal active-site of SOD is structurally located deep in a channel and direct electron

Comparison of Wild Type and Double Mutated Aequorin

transfer between enzyme and the electrode is difficult.

Variants from Luminescence and Kinetic Aspects

Keywords: Superoxide Dismutase, Superoxide Detection, Cysteine,

Mehdi Zeinoddini* 1, Khosro Khajeh 2,

Electrochemistry.

Saman Hosseinkhani 2 1. Biotechnology Research Center, Malek-Ashtar, Unversity of Technology, Tehran, IR

Abstract No.256

2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Evaluation of the Smac Based Peptides and Protein in


Antagonizing XIAP as Anticancer Agents


Saeed Balalaei 1, Jamshid Davoodi* 2, Behnaz Ahangarian Abhari 2

The

photoprotein

aequorin

is

a

small

calcium-dependent

bioluminescent protein which emits blue light by an intramolecular reaction. The emission properties, stability and decay kinetics of this

1. Dept. of Chemistry - K.N.Toosi University of Technology, Tehran, IR

reporter protein can be changed by directed mutagenesis of key


residues. In the present work, three double mutants including variants


of Y82F/W86F, Y82F/D153G, and W86F/D153G are prepared. With respect to our results, it seems that presence of W86F mutation shifts

XIAP prevents apoptosis through inhibition of caspase-9 by the BIR-3

the emission to shorter wavelengths, while the Y82F mutation results

and caspases -3 and -7 through BIR2 domain. SMAC which is released

in shift of emission to longer wavelengths. Furthermore, analysis of the

from the mitochondria competes with caspases in binding to XIAP

variants for light half-life showed decreased t1/2 for the two mutants

unleashing caspase activity and causing cell death. SMAC peptides and

of Y82F/D153G and W86F/D153G. Conversely, the Y82F/W86F variant

protein were used to investigate their ability in relieving the

displayed a 2-fold increase of light half-life compared to wild type

executioner caspase activities inhibited by both the BIR1-2 domains

aequorin. Finally, comparative thermostability analyses of double

and the BIR1-2-3 domains of XIAP. Furthermore, the potency of these

mutants showed higher stability only for Y82F/D153G variant while the

peptides was compared to the Smac protein in antagonizing XIAP.

single W86F mutant reached the highest stability against thermal

AKPD, ANPR, SGVD, AVPI peptides and the SMAC protein were

treatment. Our results suggest that replacement of few residues in the

preincubated with the IAP domains and the activity of caspases was

active site or binding pocket of aequorin affects its luminescence and

studied in the presence of these mixtures. Moreover, the ability of

kinetic properties and promises the feasibility of new reporter

these peptides in preventing the interaction of BIR1-2 domain with the

production with limited substitutions.

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Keywords: Aequorin, Site Directed Mutagenesis, Luminescence

Abstract No.259

Properties, Double Mutants. The Fibrillation Study of β-Lactoglobulin Upon Incubation with Aflatoxin M1 Abstract No.258

Mansooreh Mazaheri*, Ali Akbar Saboury, Najmeh Poursasan, Ali Akbar Moosavi Movahedi

Protective Effects of 6 Genotypes of Walnuts Against free Radical-Mediated Protein Oxidation

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IR (E-mail: [email protected])

Nahideh Tahmasebpour 1, Gholamreza Dehghan* 2, Ziba Mirzaee 1, Jafar Hajilou 3

Aflatoxin M1(AFM1) appears in milk as a direct result of the ingestion of food contaminated with aflatoxin B1 by cattle. The role of milk in

1. Department of Plant Biology, Faculty of Natural Science, University

human nutrition is well-known. The formation of AFM1 occurs in liver

of Tabriz, Tabriz, IR

and it is secreted into the milk, which is cytotoxic and genotoxic.

2. Department of Animal Biology, Faculty of Natural Science, University

AFM1 is bound to milk proteins. As a result of the binding affinity of

of Tabriz, Tabriz, IR

AFM1 for milk proteins, the toxin is distributed unevenly between whey

3. Department of Horticultural Sciences, Faculty of Agriculture,

and curd. The purpose of this report is to study the effect of AFM1 on

University of Tabriz, Tabriz, IR (E-mail: [email protected])

β-lactoglobulin (β-Lg) fibrillation.

Regards to this proposal that

AFM1 enters to whey proteins (especially, β-Lg), supposed it would interact with this protein and affects on β-Lg fibrillation. β-Lg solution

The role of oxidative protein damages in the pathophysiology of

with concentrations of 1(W/V%) at pH 2 was prepared and interacted

human diseases is currently a topic of considerable interest as oxidized

with different concentration of AFM1. After heating of the solutions at

proteins has been implicated in a wide spectrum of clinical disorders.

85 °C for 24 h, the fibrillation of them were investigated. Strange

In this study, the antioxidant activity of 6 genotypes of walnuts, were

results showed that AFM1 reduces the intensity of fluorescence of

investigated employing various established in vitro systems including

fibrils. By increasing the concentration of AFM1, the intensity of

ferric reducing ability (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH),

fluorescence of fibrils was decreased. This means AFM1 as a toxin

and inhibitory effect on protein oxidation as well as the inhibition of

reduces the fibrillation of β-lactoglobulin.

Fe2+/ascorbate induced lipid peroxidation in human plama samples. Total phenolic content (TPC) and total flavonoid content (TFC) of the

Keywords: Aflatoxin M1, β-Lactoglobulin, Fibrillation, Milk proteins.

samples were also determined by a colorimetric method. The addition of Fe2+/ascorbate to the plasma samples significantly increased the of

Abstract No.260

protein oxidation by loss of protein-bound sulphydryl (P-SH) groups and increased lipid peroxidation (LPO) The plant extracts showed

Fibrillar Protein Aggregation May be Detrimental Via

inhibitory effects against P-SH oxidation, and LPO to varying degrees.

Different Oxidative Routes: Relevance to the Etiology of

Based on this study, the protective effects of walnuts extract could be

Amyloid-Related Neurodegenerative Disorders Using

due to its TPC. In that respect, free radical induced protein oxidation

the Experimental-Based Evidences

was suppressed significantly by the addition of walnut over a range of concentration. These results clearly demonstrated that in the shells of walnut have higher antioxidant activities than the hulls of walnut. KH501, KH403, KH509 genotype with the highest phenolic content in its shells has more antioxidant activity against protein oxidation. Keywords: Protein Oxidation, Juglans Regia, Antioxidant Capacity, Lipid Peroxidation.

Reza Khodarahmi* 1, Zahra Hosseinpour 1, Sirous Ghobadi 2, Kamran Mansouri 1, Ali Mostafaie 1, Khirollah Yari 1, Seyyed Abolghasem Ghadami 1 1. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, IR 2. Department of Biology, Faculty of Sciences, Razi University, Kermanshah, IR (E-mail: [email protected]) The exact mechanism of cell death in neurodegenerative diseases remains obscure, but the aberrant assembly of proteins into fibrillar

A110


aggregates is accused to be the primary cause of pathogenesis.

analyzed. Then, the outputs of computer-designed algorithms were

Furthermore, the structural determinants of protein fibrils that are

compared with experimentally derived available information. There is

responsible for cell dysfunction are not yet clear. The main objective

the possibility that these algorithms become useful tools for screening

of the present study was to discuss the potential role of peroxidase

therapeutic approaches against amyloidoses as well as to improve the

activity of

solubility of recombinant proteins. We will discuss the importance of

“heme-amyloid fibril” complex in neurodegenerative

disorders onset/progression using the protein-based experimental

our analyses.

models, in vitro. The results of the present study also suggest that oxidative stress may be involved in neurodegenerative cell toxicity via

Keywords: Aggregation propensity, Tango, Aggrescan, Waltz.

several independent (mechanistic) routes, highlighting a possible functional link among formation of excessive amounts of reactive (oxidant) species and protein/DNA metabolism. The present findings emphasize that data on the origin of oxidative stress-related damages may help us to postpone/attenuate the onset/extent of irreversible part of neurodegenerative pathogenesis. Keywords: Amyloid Aggregation, Toxicity, Heme, Peroxidase, αCrystallin, α-Chymotrypsin.

Abstract No.262 Purification of Lipid-Transfer Protein-1 (LTP-1) from Rice Grain and Study of Drug Binding to Normal and Modified LTPs

Reza Khodarahmi* 1, Shabnam Maghsoudi 1, Mohammad Reza Ashrafi 1,2, S.Abolghasem Ghadami 1,2, Sirous Ghobadi 2, Marzieh Hamzeh 3 1. Medical Biology Research Center, Kermanshah University of Medical

Abstract No.261


Prediction of Aggregation Propensity and Amyloidogenic Regions in Proteins, Based on Their Primary Sequences

Reza Khodarahmi 1, Seyyed Abolghasem Ghadami* 1,2, Sirous Ghobadi 2 1. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, IR 2. Departments of Biology, Faculty of Sciences, Razi University, Kermanshah, IR (E-mail: [email protected]) The misfolding and extracellular amyloid depositions of specific proteins are associated with a large family of human pathologies, often called protein conformational diseases (PCDs). Despite many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. In the last decade, data have begun to accumulate suggesting that the composition and the primary structure of a polypeptide determine to a large extent its propensity to aggregate and that small changes may have a huge impact on solubility and stability. The ability of identification of individual amyloidogenic regions in disease-linked polypeptides would be of much value. In this study, “aggregation-prone” sequences in 2 native/modified nondisease related amyloidogenic proteins, calculated

2. Departments of Biology, Faculty of Sciences, Razi University, Kermanshah, IR 3. Departments of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, IR (E-mail: [email protected]) Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins which transport phospholipids between membranes and are subdivided into two subfamilies, nsLTP-1 (9 kDa) and nsLTP-2 (7 kDa) according to the molecular weight. All of nsLTPs are highly stable proteins because they possess eight highly conserved cysteine residues forming four disulfide bonds. These proteins have received an increasing interest as potential drug carriers in drug delivery systems. These highly stable proteins have potential to protect drugs against oxidation or degradation. In the present study, citraconylation was employed to modify the accessible lysine residues of LTP-1. Then amphotericin B, which is widely used as an antifungal drug, has been used to compare the drug binding behaviors of the native and modified proteins. Fluorescence spectroscopy was used to compare the thermodynamics as well as PSH properties of native and modified LTP1. Our results showed that Kbinding, the number of binding site and PSH have been increased in modified LTP. We may propose that new binding sites have been created upon LTP modification or binding ability of protein to drug has increased compared to the native one. We will discuss the importance of our observations. Keywords: LTP, Drug, Modification.

by three prediction servers (Tango, Aggrescan and Waltz) were

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Abstract No.264

Abstract No.263 Comparative Evaluation of Sildenafil Effects on the

Differential Binding and Effect of Leucovorin on Stability and

Structure and Activity of Native and Modified States of

Aggregation of IgG

Human Carbonic Anhydrase II

Rizwan Khan* 1, Ejaz Ahmad 1, Sumit Chaturvedi 1, Gulam Rabbani 1, Reza Khodarahmi

1,2

1

Abhay Singh 1, Vaishali Kapoor 2, Sharmistha Dey 2, Satya Das

, Nooshin Bijari* , 3

Seyyed Abolghasem Ghadami , Sirous Ghobadi

2

3

1. Interdisciplinary Biotechnology Unit, Aligarh Muslim University 1. Medical Biology Research Center, Kermanshah University of Medical

Aligarh – 202002, IN


2. Department of Biotechnology, All India Institute of Medical Sciences,

2. Departments of Pharmacognosy and Biotechnology, Faculty of

New Delhi 110029, IN

Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, IR


3. Departments of Biology, Faculty of Sciences, Razi University, Kermanshah, IR

The interaction of leucovorin (LV), an adjuvant used in cancer


chemotherapy, with a non-carrier protein IgG was performed by different spectroscopic and computational approaches in absence and

Human carbonic anhydrase (hCA, EC 4. 2. 1. 1), is a zinc – containing

presence of physiological blood solutes (NaCl, urea and glucose) as

enzyme which catalyes the reversible hydration of carbon dioxide to

well as in those conditions where their concentrations increases

bicarbonate and hydrogen ions. Many of CA isozymes have been

abruptly (in diabetes and uremia). A continuous decrease in Stern-

discovered as important targets for activators and inhibitors with

Volmer fluorescence quenching constants with the increase in

clinical applications. It has recently been demonstrated that sildenafil,

temperature (from 25 to 37°C) and a higher value of bimolecular rate

which widely used for the treatment of erectile dysfunction, acts as

constant showed static mode of fluorescence quenching. The specific

activator of hCA II. We observed that histidine residues on the rim of

nature of LV binding with a non-carrier protein is confirmed by SPR.

hCA II active site are critical for its activity and suggestive of their role

For this interaction, the enthalpy (∆H) and the entropy (∆S) changes

as a proton transfer group. The treatment of histidine-modified hCA II

were found to be 6.46 kcal mol-1 and 45 cal K-1 mol-1 respectively along

with sildenafil revealed a moderate hCA II activation profile.

with negative signs of free energy change (∆G) which suggest that

Furthermore, the effects of sildenafil on kinetic and structural

hydrophobic interaction is the predominant intermolecular forces

properties of native and modified hCA II were investigated employing

stabilizing the complex. The binding results suggested that the main

different spectroscopic techniques such as UV-Vis, circular dichroism

responsible driving forces are H-bonds, hydrophobic and electrostatic

(CD) and fluorescence spectroscopy. Fluorescence data proposed that

interactions which are consistent with docking results. We have

sildenafil acts as quencher of native and modified hCA II fluorescence.

postulated that both the uremic and diabetic patients are at higher risk

Both the Stern-Volmer analysis and molecular docking revealed the

regarding the bioavailability of required amount of drug. We have also

existence of one binding site in the both forms of enzymes for

investigated the effect of LV on IgG conformation accompanying the

sildenafil. The thermodynamic parameters indicated that the driving

change in protein stability as well as the amyloidogenic propensity of

force of this processes are not same. Calculation of the protein surface

IgG in the presence and absence of normal and increased level of the

hydrophobicity (PSH), using 1- anilinonaphthalene-8-sulfonic acid

blood solutes. The plausible effect of LV on IgG was to enhance the

(ANS), indicate the increment of PSH of native and modified hCA II in

stability and somehow to diminish the aggregation of IgG molecules.

the presence of sildenafil. The near-UV CD results as well as the determination of PSH results reiterate that, in the presence of

Keywords: Disease Mimetic Conditions, Bioinformatics, Non-Carrier

sildenafil, flexibility of the native and modified hCA II tertiary structures

Protein, Protein Aggregation, Protein-Ligand Interaction, Protein

has been increased. We will discuss the importance of our

Stability, Thermodynamics.

observations. Keywords: Human Cabonic AnhydraseII, Sildenafil, Fluorescence Quenching, Activator, Diethyl Pyrocarbonate, Histidine Residue.

A112


Abstract No.266

Abstract No.265 The Physicochemical and Biochemical Survey of the

40 Years of Protein Biochemistry with Escapades

Honey Types from Iran: Protein and Enzyme Profiling

Bernhard Erni* Elahe Mahmoodi Khaledi* 1, Mehran Habibi-Rezaei 1, Nasim Kashef 2, Iisa Sadeghian 1

Department for Chemistry and Biochemistry, Universität Bern, CH (E-mail: [email protected])

1. Protein Biotechnology Research Lab (PBRL), School of Biology, College of Science, University of Tehran, Tehran, IR

Biochemistry covers a vast area, and I was fortunate to visit a number

2. Medical Bacteriology Lab ,School of Biology, College of Science,

of spots in it. Membranes caught my attention already when I was a

University of Tehran,Tehran, IR

student. In my first encounter during my diploma thesis I calculated the


vibrational

modes

of

different

triglyceride

esterbackbone

conformations. The purpose was to characterize the "real" backbone Honey has been used since ancient time as both a sweet feed and

conformation from the infrared spectra of lipid multilayers. I then

a traditional medical

The

switched the topic for my PhD, where I helped to establish an in vitro

greatest biochemical fraction of the honey weight is pertains to the

system of mammalian protein synthesis with purified components. At

sugars and their sugar profile are applied to characterize the honey

that time such a system was deemed necessary to elucidate the

quality the origin of honey. Moreover, the acidity of honey is one of the

possible role of protein synthesis in the generation of antibody

most important factors in quality control of honey. Over time, simple

diversity. As a postdoctoral fellow - back on the main track - I

sugars and acids present in honey provides

to

synthesized a photoaffinity label for the analysis of apolar membrane

produce the hydroxyl methyl furfural (HMF), therefore its quantity is an

lipid-protein interactions. Unfortunately, the label in the apolar

important but small percentage of the honey weight. factor in

environment reacted by intramolecular rearrangement instead of

controlling

crosslinking and thus was useless. Benefitting from my experience in

of

treatment of

honey

many

diseases.

condition

freshness.

Proteins

are

the

most important constitute however, at 0.1-0.5% of the honey weight.

protein purification I then started to purify the glucose transporter of

This

plant origin,

the Escherichia coli phosphotransferase system (PTS), the protein that

and also diverse species of bees. Determination of protein content of

became the golden thread of all subsequent research. One reason to

honey is considered a new method for evaluating honey quality. In this

choose this protein and no other was the fact, that it also has

study, the physicochemical properties and protein contents and

phosphotransferase (kinase) activity that is much easier to assay in

enzyme profiling of 20 honey samples collected from different region of

vitro than transport. Along the way of cloning the permease gene a

Iran were investigated. In this survey physicochemical parameters

second PTS transporter was picked up, which appeared even more

such as pH, acidity, ash, HMF and sugar content of samples were

interesting because it also serves as membrane gate for the

determined and compared. Upon extraction and concentration by

penetration of bacteriophage DNA and of pore-forming bacteriocins

dialysis, centrifuge and ammonium sulfate precipitation methods, the

(toxic peptides). Gene sequencing took almost two years, an effort that

protein content were detected by Bradford assay. The result of this

payed off because it allowed us to overexpress the proteins, to purify

study showed that the physicochemical parameters and protein

them by metal chelate chromatography and to study their function by

contents of honey types were different. These data can be used in

site-directed as well as random mutagenesis. Because PTS occur and

determination of honey quality and its therapeutic features and provide

play an important role only in bacteria, it appeared that it could be a

beneficial information for future studies in this field.

target of antibacterial agents. This perspective motivated us to develop

amount

differs

according

to

the

in vivo and in vitro assays for high throughput inhibitor screening, to Keywords: Honey, Quality Control, Physicochemical Properties,

characterize the attenuated virulence of a PTS knock-out strain, and to

Protein Content.

solve the X-ray structures of several soluble PTS protein subunits. Some of the projects were done in collaboration with a start-up company. Proteome analysis of the knock-out strain then draw our attention to a new family of dihydroxyacetone kinases. They are homologous to the eucaryotic Dha kinases but are supplied with high energy phosphate by the PTS rather than by ATP. The elucidation of their structure, catalytic mechanism and gene expression were the

A113


conclusion of 40 exciting years spent in the biochemistry laboratories

Abstract No.268

of different countries with various cultures. Interaction of Cationic Peptide Drugs with Bacteria and Lipid Keywords:

Protein

Biochemistry,

Backbone

Conformation,

Bilayers: Short R-, W-Rich Hexapeptides as a Case Study

Phosphotransferase System, Phosphotransferase, Proteome Analysis.

Mojtaba Bagheri* Department of Chemical Biology, Leibniz Institute of Molecular

Abstract No.267

Pharmacology, Berlin, DE (E-mail: [email protected])

Effect of Butachlor Herbicide on Hemoglobin Structure and Species

The global emergence of resistance to antimicrobial agents is increasingly limiting the effectiveness of current drugs. The treatment of

Leila Fotouhi*, Ali Akbar Saboury, Ali Akbar Moosavi-Movahedi

multidrug-resistant

Gram-negative

germs

represents

a

particular

challenge. Antimicrobial peptides are effective molecules in the innate immune system and might provide a promising alternative towards

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IR

classical antibiotics. Understanding the structural basis of activity and


bacterial selectivity provides one basis for the development of effective peptide-based antimicrobial drugs. Peptides rich in arginine (R) and

Butachlor (2-chloro-2', 6‟-diethyl-N-(butoxymethyl) acetanilide) is a

tryptophan (W) residues are of particular interest as they are found as

member of chloroacetanilide class of chemistry and is the herbicidal

small antimicrobial motifs in much larger natural compounds. Our recent

active ingredient in MACHETER EC. This herbicide is used as a pre-

strategy to induce constraints in RW-rich hexapeptides by cyclization did

emergence control for the undesirable grasses and broadleaf weeds

result

.The consumption of butachlor in Iran is among the most used

Escherichia coli, for instance, cyclo-RRRWFW (c-WFW). The activity of c-

pesticides which is mostly applied to rice fields. Extensive use of this

WFW against E. coli is modulated by lipopolysaccharides (LPS) in the

herbicide beside other types of pesticides is now a concern for human

outer bacterial membrane.To elucidate the role of the two tryptophan

health, as these chemicals can enters the body through our foods.

residues in interactions with E. coli membranes, we replaced them by

Although most of these pesticides and their metabolites are excreted

analogues having altered hydrophobicity, dipole and quadrupole

from the body, high daily intake cause permanent existence of these

moments, hydrogen-bonding ability, amphipathicity, or ring size. The

pesticides in the body. As a consequence, entrance of this herbicide

biological activitiy against Bacillus subtilis and erythrocytes increased with

into the blood stream, brings one of most abundant blood protein,

increasing peptide hydrophobicity, whereas the effect on E. coli revealed

hemoglobin, into contact with butachlor which may manipulate

a more complex pattern. Isothermal titration calorimetry demonstrated

hemoglobin function in the body. In this report, the interaction of

that peptide partitioning into model lipid membranes is driven by both

hemoglobin with butachlor under physiological condition was assessed

electrostatic

and found the changes in protein structure and function which was

order: POPC/smooth-LPS >> POPC/rough-

analyzed by various methods such as UV-Vis, fluorescence as well as

LPS > POPC/lipid A = POPC/POPG > POPC.

biophysical and biochemical investigations. The hemoglobin species

contributions to binding to POPC and mixed POPC/POPG were

upon interaction of butachlor were studied in this report.

comparable. Low hydrophobicity and peptide conformational flexibility

in

pronounced

and

peptide

hydrophobic

activity

against

interactions

and The

Gram

negative

follows

the

hydrophobic

reduced binding, showing that peptide–membrane interactions correlate Keywords: Herbicide, Butachlor, Hemoglobin, Spectroscopy.

with the biological effect. The highly differentiated activity pattern against E. coli was poorly reflected in peptide binding to POPC/lipid A and disappeared in studies with LPS-containing membranes. Stronger partitioning into POPC/smooth-LPS as compared with POPC/r-LPS uncovered a significant role of O-antigen and outer-core oligosaccharides of LPS in anti-E. coli activity. Keywords:

Cationic

Lipopolysaccharide, Calorimetry.

A114

Antimicrobial

Peptide

Peptides,

Partitioning,

Lipid

Bilayers,

Isothermal

Titration


Abstract No.269

Abstract No.270

Understanding the Role of Conserved Residues in Folding

A Hydrogen Peroxide Biosensor Using Functionalized-Carbon

and Stability of Cytochromes-C

Nanotubes and Clay

Faizan Ahmad*

Farideh Gouranlou* 1, Hedayatollah Ghourchian 2

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia

1. Biophysic, Institute of Biochemistry and Biophysics , University of

Islamia, New Delhi 110 025, IN

Tehran, IR


2. Laboratory of Microanalysis, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IR

There are more than 287sequences of cytochromes-c (cyts-c) known


(http://pir.georgetown.edu/). 37 of them are of mammalian origin. All the mammalian mitochondrial cyts-c are ~104-residue long single

Among the different analytical devices, biosensors play an important

polypeptide chain in which the heme is covalently linked through Cys14

role due to some generally claimed advantages: intrinsic specificity,

and Cys17. A sequence alignment of cyts-c from all kingdoms with that

low costs and fast analyses. . In this study, we demonstrate the

of the horse cyt-c led to the conclusion that there are only 5 positions

biosensor based on deposition of carbon nanotubes (CNTs) on clay

(Cys17, Gly29, Gly41, Leu68, Pro71) which are conserved throughout the

minerals, and the development of biosensors based on COOH–

kingdoms. Interestingly, all mammalian cyts-c have Leu at position 94

MWCNT/Clay/horse radish peroxidase (HRP) for the detection of

that, baring 13 species which have either Ile or Val or Phe at this

hydrogen peroxide (H2O2). The mixed hybrid film of CNT/Clay/HRP was

position, is conserved throughout the kingdom. Such conserved residues are sometimes also called as ‘key residues’. We have been interested in

coated on the glassy carbon (GC) electrode. This film exhibited a detection limit of 5.0 × 10− 5 M towards H2O2 with a sensitivity of

understanding the role of this key residue (Leu94) of the mammalian

280 µA mM− 1. A higher sensitivity and more stability of enzyme are

cyts-c in protein folding and stability. We present here the results of

observed with increasing H2O2 content in the composite matrix film.

mutation of Leu by Gly at the position 94 of the horse cyt-c whose 3-D

Consequently, the CNT/Clay medium can probably be a useful

structure is known. In silico study shows that this mutation (Leu94Gly)

electrode for the development of sensors due to its high sensitivity and

will lead to removal of 10 van der Waals interactions. This study

applicability.

therefore suggests that the mutant Leu94Gly will be less stable than the wild-type protein. We prepared the mutant Leu94Gly by expressing it in

Keywords:

E. collie, and we carried out in vitro studies of structural and

Peroxidase, COOH Functionalized -Carbon Nanotubes, Hydrogen

Carbon

Annotates,

Clay,

Biosensor,

Horse

Radish

thermodynamic characterizations. Our main findings from the in vitro

Peroxide.

studies are: (a) the mutant protein exists as molten globule under the native condition of the wild-type protein, (b) a weak salt denaturant induces a biphasic transition in which the equilibrium intermediate has structural characteristics of the pre-molten globule, (c) the mutant leu94Gly is unfolded at pH 2, and titration of the this unfolded protein with NaCl also induces a pre-molten globule state, and (d) Tm (thermodynamic stability) and ∆GDº (thermodynamic stability) of the mutant are respectively, 29 ºC and 5 kcal mole-1 less than those of the wild-type protein. A comparison these results with those of the wild-type protein led us to conclude that the conserved residue Leu94 in the wildtype protein is required for proper protein folding and stability. The

Abstract No.271 Spectrophotometery Studies on the Interaction of Au(III)(Phend)Cl3 new Complex with Calf Thymus DNA

Sasan Moradi* 1, Davood Ajloo 1,2, Taghi Lashkarbolouki 3, Robabeh Alizadeh 1, Mina Evini 4, Ali Akbar Saboury 4, Ali Akbar Moosavi-Movahedi 4 1. School of Chemistry, Damghan University, Damghan, IR

mechanism of folding of cyts-c may be described by the process,

2. School of Biology, Damghan University, Damghan, IR

Unfolded state ↔ Pre-molten globule state ↔ Molten globule state ↔

3. Institute of Biological Science, Damghan University, Damghan, IR

Native folded state under physiological condition.


Keywords:

Conserved

Cytochromes-C.

Residue

in

Protein,

Folding,

Protein,

(E-mail: [email protected]) Interaction between calf thymus DNA and (Phend)Cl3-Au(ІІІ) new complex in physiological buffer (pH=7.2) was investigated using UV-Vis

A115


cyclic

is not entirely at the transcriptional level, suggesting possible points of

voltammetry and viscosity measurements. The decrease of the

post-transcriptioal thermal sensitivity, so our direct application of

absorption spectra of (Phend)Cl3-Au(ІІІ) complex were observed in the

hyperthermia on macrophages without any treatment supports this

presence of DNA, and the fluorescence intensity of (Phend)Cl3-Au(ІІІ)

hypothesis. The data in this study reveal the potential of mild

was decreased with the addition of DNA. The relative viscosity of DNA

hyperthermia (elevated physiological temperature) to increase No.

increased with the addition of (Phend)Cl3-Au(ІІІ) complex. The

production and iNOS synthesis in using peritoneal type macrophage

calculated binding constants of (Phend)Cl3-Au(ІІІ) complex with DNA

like cell line. These may support the concept that altering the thermal

at 250 nm and 293 K were 5×106 M-1. Cyclic voltamograms due to

micro environmental would be an important means by which the host

cyclic voltammetry showed that, cathodic peaks shifted to positive

can affect the macrophage responses.

spectrophotometery,

fluorescence

spectrophotometry,

potential that indicates intercalation interaction between (Phend)Cl3Au(ІІІ) complex and CT-DNA. All these results indicated that

Keywords: Nitric Oxide, Nitric Oxide Synthase (iNOS), Macrophage,

(Phend)Cl3-Au(ІІІ) complex can bind to DNA and the major binding

IC-21 Cell Line and Hyperthermia.

mode is intercalative binding. Keywords:

Calf-thymus

DNA,

(Phend)Cl3-Au(ІІІ),

Interaction,

Abstract No.273

Spectrophotometry, Intercalation. Detection of Fibrillar Aggregates and Inhibition of AmyloidMediated Peroxidase Activity Using the Novel BenzothiazoleAbstract No.272

and Benzofuranone-Derivatives Fluorescence Compounds

Hyperthermia Effects on IC-21 Macrophage Like Cell Line To

Seyyed Abolghasem Ghadami 1,2, Reza Khodarahmi* 1,3, Hadi Adibi 3,

Assay Activity and Expression of Inducible Nitric Oxide

Sirous Ghobadi 2

Synthase (iNOS) Enzyme 1. Medical Biology Research Center, Kermanshah University of Medical

Manoochehr Goojai 1, Samideh Khoei 2, Bahram Goliaei* 2

Sciences, Kermanshah, IR 2. Departments of Biology, Faculty of Sciences, Razi University,

1. Department of Biology, Azad University of Bonab, East Azerbayjan, IR

Kermanshah, IR

2. Laboratory of Biophysics and Molecular Biology, IR

3. Faculty of Pharmacy, Kermanshah University of Medical Sciences,


Kermanshah, IR (E-mail: [email protected])

Nitric oxide (No.) is a uniquely diffusible and reactive molecular messenger in vascular and immune systems, motivated researches for

Alzheimer’s disease is characterized by the presence of amyloid

evaluating its biosynthesis by the macrophages. As macrophages are

deposition. Thioflavin T (ThT) has been one of the molecules of choice

often called to function at times of elevated ambient temperature (e.g.

to attempt the detection of amyloid deposits, however, it has been

during local inflammation or systemic fever), it is possible that their

reported that ThT was unable to cross blood–brain barrier (BBB). Since

production of critical effector molecules such as nitric oxide ion or

compounds without a permanent positive charge are mainly capable of

nitric oxide synthase (iNOS) , is sensitive to physiological changes in

crossing the blood–brain barrier, there is a strong motivation to

temperature. To test this possibility, the threshold requirement for

develop suitable compounds for in vitro fibril quantification as well as

production of No. and iNOS in macrophage like cell line of IC-21 under

for in vivo amyloid imaging. Moreover, oxidative stress has frequently

normothermic conditions (37 ˚C) and following mild hyperthermia (40,

been reported to play a critical role in the onset/progression of some

42 and 44 ˚C) were compared. Temperature gradient showed

neurodegenerative disorders. In this study, we synthesized and

considerable increases of No. concentration at 40 and 42 ˚C and

employed benzothiazole and benzofuranone derivatives (including

decrease at 44 ˚C (24 h incubation).

Further, if IFN- and

neutral ThT analogues), both as fluorescent probes to quantitatively

lipopolysaccharide (LPS) were given before thermal exposure, a

determine the amyloid fibrils made of chymotrypsin (and crystalline)

substantial increase in No. and iNOS was observed (highest at 42 ˚C

and as potential inhibitors for peroxidase activity. Analyses of the in

after 24 h incubation) over that seen using cells kept exposed to

vitro binding studies indicated that compounds 2 and 4 bind to the

hyperthermia alone or that of at normthermic conditions (37 ˚C). As

amyloid structures successfully while compounds 1 and 3 showed a low

RT-PCR data have revealed the thermal regulation of iNOS expression

affinity in binding to fibrils. Furthermore, compounds 3 and 4 were

A116


observed to inhibit amyloid-mediated peroxidase activity in a reversible

interaction with glibenclamide which was in good agreement with the

un-competitive manner. The resulting data may be useful in providing

theoretical analyses.

mechanistic insights to develop potential diagnostic, curative, and/or preventive

strategies

in

vivo

against

amyloid-related

neurodegenerative disorders. We will discuss importance of our

Keywords:

Human

Carbonic

Anhydrase

II,

Glybenclamide,

Competitive Inhibition, Binding Study.

observations. Keywords: Amyloid Detection, Benzothiazole And Benzofuranone,

Abstract No.275

Peroxidase Activity. Effect of Boswollia Extract on Dynamicity of Microtubule Protein, Related Memory and Consciuousness Abstract No.274

Gholam Hossein Riazi*, Dayhim Atarod, Ghazaleh Eskandari sedighi, Ali Afrasyabi, Seyed Morteza Karimi

Investigation on the Effects of Glibenclamide on the Structure and Function of Human Carbonic Anhydrase ІІ

Institute of Biochemistry and Biophysics, University of Tehran, IR

Sirous Ghobadi 1, Reza Khodarahmi 2,3, Mona Pazhohi* 1,


S. Abolghasem Ghadami 1, Noushin Bijari 1 Tubulin and microtubule protein have been defined as constants of 1. Department of Biology, Faculty of Science, Razi University,

neural processes result8ing memory and consciousness. Microtubule

Kermanshah, IR

protein is composed of tubulin dimmers α and β. Microtubule Polymer


(MTP) is a dynamic polymer residing mostly in neural axon and


dendrites as well as mitotic spindle. It has been shown that decreasing

3. Faculty of Pharmacy, Kermanshah University of Medical Sciences,

polymerization rate or shortening of MTP results in decreasing memory

Kermanshah, IR (E-mail: [email protected])

and

consciousness-based

memory.

Therefore

deformation

or

deactivation of MTP has been shown in Alzheimer’s disease. Lots of effort have been conducted to enhance polymerization for memory loss

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which

remedy.In our study Boewollic acid was extracted from Boswollia gum.

catalyze the reversible hydration of CO2 to form bicarbonate and

Different doses of Boswollic acid and taxol were prepared and tested

proton. CAs inhibition is exploited clinically for decades for various

on polymerization of tubulin at 37˚c and at the presence of 1mM GTP.

classes of diuretics and systemically acting antiglaucoma agents. In the

In spite of the fact that both compounds increased the length of the

last years, novel applications of CA inhibitors emerged, such as

tubuline polymers, taxol inhibited dynamicity of microtubule proteins,

topically acting anticonvulsants, antiobesity, antipain, and antitumor

while Boswollic acid increased the dynamicity. In vivo studies showed

agents/diagnostic tools. In this study, we used the combination of

that taxol decrease memory of albino mouse while Boswollic acid

computer modeling with spectroscopic techniques, such as UV-Vis,

enhanced the memory and consciousness of animal by several factor.

fluorescence and circular dichroism (CD) spectroscopy to investigate

We suggest substituted drugs from Boswollia extract for enhancing

the effects of glibenclamide, a sulphonylurea drug, on the human

neuroplasticity.

carbonic anhydrase II (hCA II) structure and function. Kinetic studies showed that glibenclamide inhibits hCA II esterase activity via a simple

Keywords: Microtubule Protein, Consciuousness, Tubulin, Neural

competitive mode. Stern-Volmer analysis of quenching data at different

Axon.

temperatures revealed that the intrinsic fluorescence of enzyme was quenched through a dynamic quenching mechanism. Analysis of the thermodynamic parameters of binding showed that hydrophobic interactions play the major role in stabilization of the enzyme–drug complex.The results of the surface hydrophobicity index determination, chemical modification of the surface tryptophans and CD spectroscopy showed occurrence of some compactness in hCA II structure due to

A117


Abstract No.277

Abstract No.276

Targeting Mesothelin for Treatment of Cancer

Biophysical Targeting of OmpF Nano-Channel Forming Proteins, Translocating Ofloxacin Antibiotic into E.Coli,

Kun Wang 1, Vidya Bodempudi 1, Zhengian Liu 1, Emma Borrego-Diaz 1,

Involved In Urinary Tract Infection

Farnaz Yamoutpoor 1, Weihong Pan 1, Arkadiusz Z. Dudek 1, 1

Maryam Rezaei* , Hamid Mobasheri

2

Mojtaba S. Olyaee 1, Tuba Esfandyari 2, Faris Farassati* 3

1. Institute of biochemistry and biophysics (IBB), IR

1. The University of Kansas Medical Center, Department of Medicine,

2. Lab. Mem. Biophysics, Institute of Biochemistry and Biophysics,

Division of Gastroenterology, Hepatology and Motility, Molecular

University of Tehran, IR

Medicine Laboratory, Kansas City, Kansas, US


2. Department of Internal Medicine-Molecular Medicine Laboratory, Divisions of Gastroenterology and, Hematology/Oncology, Kansas, US

OmpF is an ion channel in outer membrane of E.coli that is responsible


for translocation of molecules with an exclusion limit of 600Da. These

Divisions of Gastroenterology and Hematology/Oncology, Molecular

channels are known for the path they form for translocation of


hydrophilic

molecules, antibiotics,

needed

to

pass through

the


hydrophobic membrane. An efficient antibiotic is expected to exhibit high affinity for certain target cell and molecules and reach them

Mesothelin, a differentiation antigen present in a series ofmalignancies

rapidly. Quinolons and Beta-lactames are among the successful and

such as mesothelioma, ovarian, lung and pancreaticcancer, has been

widely used antibiotics known to cure bacterial infections at present.

studied as a marker for diagnosis and a target forimmunotherapy. We,

The antibiotic that enter the bacteria kill them by inhibiting DNA gyrase

however, were interested in evaluating theeffects of direct targeting of

or the topoisomerase type 2 enzymes as a target, thereby interfering

Mesothelin on the viability of cancercells as the first step towards

the DNA replication and translocationIn this work, we investigated the

developing a novel therapeuticstrategy. We report here that gene

passage of Ofloxacin antibiotic through the single OmpF porin of E.coli

specific silencing for Mesothelinby distinct methods (siRNA and miRNA)

by means of voltage clamp technique in real time to address the drug

decreased viability ofcancer cells from different origins such as

trafficking in urinary tract infection disease in human. We also

mesothelioma (H2373),ovarian cancer (Skov3 and Ovcar-5) and

investigated the effect of the passing antibiotic on channel gating,

pancreatic cancer(Miapaca2 and Panc-1). Additionally, the invasiveness

conductance, and voltage sensitivity. The experimental work was

of cancercells was also significantly decreased upon such treatment.

further elaborated by theoretical and modeling approaches to address

We theninvestigated pro-oncogenic signaling characteristics of cells

the exact electrostatic effect of antibiotic on the lining group in

uponmesothelin-silencing

particular those at eyelet area of the nano-channel that constrict it to

inphospho-ERK1 and PI3K/AKT activity. The molecular mechanism

a diameter of about 0.4nm. Our results showed that the Ofloxacin is

ofreduced invasiveness was connected to the reduced expression ofβ-

not soluble in lipid phase and did not pass through the membrane.

Catenin,

Although the voltage sensitivity of the channel was not changed in the

mesenchymaltransition). Ero1, a protein involved in clearing unfolded

presence of the antibiotics, the frequency of gating increased and fast

proteins anda member of the ER-Stress (endoplasmic reticulum-stress)

flickering

pathwaywas

was

caused

due

to the

transient

obstruction.

The

an

also

which

important

markedly

revealed

marker

reduced.

a

of

significant

EMT

decrease

(epithelial-

Furthermore,

Mesothelin

conductance of the channel also remained the same indicating lack of

silencingcaused a significant increase in fraction of cancer cells in S-

any binding and conformation induction caused by the antibiotic.

phase.In next step, treatment of ovarian cancer cells (OVca429) with alentivirus expressing anti-mesothelin microRNA resulted insignificant

Keywords:

OmpF Channel, Ofloxacin Antibiotic, Urinary Tract

Infection, Translocation.

loss of viability, invasiveness, and morphologicalalterations. Therefore, we propose the inhibition of Mesothelin as apotential novel strategy for targeting human malignancies. Keywords: Signaling.

A118

Mesothelin,

Cancer,

Immunotherapy,

Pro-oncogenic



Abstract No.278

Division of Gastroenterology, Hepatology and Motility, Molecular Molecular Dynamic and Docking Study on to Interact Tetra


Sodium Sulphunated Nickel (ІІ) Phthalocyanine (NiPcTS)

2. Department of Internal Medicine-Molecular Medicine Laboratory,

and Adenosine Deaminase

Divisions of Gastroenterology and, Hematology/Oncology, Kansas, US

Seyyed Morteza Fazeli* 1, Davood Ajloo 1,2

Medicine,Divisions of Gastroenterology and Hematology/Oncology,

3. The University of Kansas Medical Center, Department of Molecular Medicine Laboratory, Kansas City, Kansas, US (E-mail: [email protected])

1. School of Chemistry, Damghan University, Damghan, IR 2. Institute of Biological Science, Damghan University, Damghan, IR (E-mail: [email protected])

Ral

(Ras

like)

leads

pathway,downs-stream

an of

important Ras,

proto-oncogenic

involved

in

multiple

signaling facets of

All-atom molecular dynamics simulation of adenosine deaminase (ADA)

neoplastictransformation. Since overactivation of Ras is a prevalent

was studied in the absence and presence of 0.014, 0.028 and 0.042 M

molecularabnormality in hepatocellular carcinoma (HCC), we decided

of (NiPcTS) and different temperatures by Gromacs (4.5.4) molecular

to study therole of RalA activation in human cancers. For example,

dynamics. One molecule of adenosine deaminase including 349 amino

RalA was found tobe significantly overactivated in HCC cell lines as well

acid immerged in a box with 9.105×9.105×8.660 nm3. Then, different

as HCC tissues.Other elements of RalA pathway, such as Ral binding

number of (NiPcTS) added to the cited. After preparing the input files,

protein (RalBP1)and Ral guanine nucleotide dissociation stimulator

the system optimized at 275, 300, 325, 335, 350, 365, 375, 390, 425,

(RalGDS), wereexpressed at higher levels in malignant samples. Aurora

and 450 K in the 20000 ps timescale. Accessible surface area (ASA),

kinase, anupstream activator of RalA, was also found to be more

circular dichroism at 222nm (CD222nm), mid-point of transition

phosphorylated(activated) in HCC as compared with non-malignant

temperature (Tm), number and distance of hydrogen bond, radial

samples.

distribution function and other physical parameters were got from

regulator of Ral activation,was expressed at comparable levels.

analysing trajectory of molecular dynamics. Results of calculated heat

Inhibition of RalA by gene- specificsilencing caused a robust decrease

capacity at constant pressure (Cp) showed that transition temperature

in the viability and invasiveness ofHCC cell lines. Additionally, the use

increases by increasing the (NiPcTS) concentration. Mid-point of

of geranyl-geranyl transferaseinhibitor (GGTI, an inhibitor of Ral

temperature transition (Tm) got as 350 and 365 K in the absence and

activation) and Aurora kinase inhibitor IIresulted in a significant

presence of 0.014 M of (NiPcTS), respectively. Thus two peaks will be

decrease in the proliferation rate of HCC cells.Furthermore, we

viewed in the plot of Cp versus temperature. Radial distribution

investigated the levels of RalA activation in HCC stemcells and

functions show that (NiPcTS) at low concentration behaves same as

concluded that active RalA, Ras and phospho-Aurora kinaselevels were

osmolytes that increases the beta form, increases the stability. Docking

increased in the fraction of cells which express CD133, awidely

energy showed that binding energy of metalic is lower than nonmetalic

accepted marker for cancer stem cells. Investigation of the level ofRalA

phthalocyanine.

activation in transgenic mouse model for HCC (FXR-Knockout)revealed

However,protein

phosphatase

2A

(PP2A),

a

negative

an elevated level of RalA-GTP in the liver tumors as compared toliver Keywords: Docking, Heat Capacity, Thermal Stability, Transition

tissue

Temperature.

modelfor HCC confirmed effectiveness

from

background

animals.

Finally,

subcutaneous mouse

of inhibition of

Aurora

kinase/Ral pathwayin reducing the tumorigenesis of HCC cells in vivo. In conclusion, RalAoveractivation is portrayed by our investigations to be an importantdeterminant of malignant phenotype in differentiated

Abstract No.279

and stem cells ofHCC. Resembling data was obtained by our team RalA as a Therapeutic Target in Human Malignancies

studying othermalignancies such as cancers of lung, ovary and

and their Cancer Stem Cells

neurological system.Therefore, intervention with this signaling pathway has the potential tooffer a novel strategy for the treatment of a

Mohamad Ezzeldin 1, Emma Borrego-Diaz 1, Mohammad Taha 1,

number of important human cancers.

Kristina Lialyte 1, Amanda Wise 1, Grace Guo 1, Kun Wang 1, Stephen Williamson 1, Tuba Esfandyari* 2, Mojtaba Olyaee 1, Faris Farassati

Keywords: RalA, Malignancies, Cancer Stem Cells, CD133.

3

A119


Abstract No.281

Abstract No.280 Homocysteinylation of Intrinsically Disordered Proteins (IDP)

MesoScopic Biophysics – The Foundations, Frontiers & The

and their Tranformations

Challenges

Thomas Haertlé*

Masroor Hassan Shah Bukhari*

Fonctions et Interactions des Protéines, BIA, Inst.Ntl Res.

Higher Education Commission of Pakistan at The NED University of

Agronomique, Nantes, FR

Engineering and, Technology, Department of Biomedical Engineering,


Stadium Road, Karachi, PK (E-mail: [email protected])

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human

Physics at the interface of microscopic and nanoscopic scales, the so-

pathologies. However, the role of homocysteinylation of lysyl residues

called “Mesoscopic Scale”, ranging from 0.1 to 1.0m, has immense

is still poorly known. In order to study the structural impact of

potential for the understanding of living systems, especially in cellular

homocysteinylation of IUPs intrinsically unstructured proteins such as

physiology. This is the regime where classical effects tend to drop off

ovine PrP and bovine caseins were used. Besides ovine PrP, αS1-, β-

and the quantum mechanical effects begin to affect the dynamics of

and κ-caseins, showing different aggregations and micelle formation,

the system. This talk presents an acquaintance with the interesting

were modified with homocysteine-thiolactone. Intrinsic and extrinsic

biophysics at the mesoscopic scale. Starting with a quick orientation

fluorescent markers such as Trp, thioflavin T, ANS and CD spectra,

with underlying foundations of the subject, a discussion on various

reveal structural changes of

casein and PrP structures after

techniques and problems in mesoscopic biophysics is presented. The

homocysteinylation reflected by an increase in beta-sheet content

talk builds an argument by developing a rationale that how could

characteristic of amyloid-like transformations. Homocysteinylation of

physics at the mesoscopic scale be different from that at the nano and

studied IUPs leads in all cases to aggregation. The sizes of aggregates

micro scales and the way it could possibly have significant

and aggregation rates were dependent on homocysteine thiolactone

repercussions on living systems. The possibilities of emergence of

concentration and temperature. N-Hcy-PrP formed insoluble multimers.

biophotons within the Infra-red spectral regions, photon evanescent

DLS and microscopic studies of modified caseins and PrP have revealed

tunneling and Quantum Mechanical Resonant Tunnelling (QMRT) of

the formation of large aggregates of about 1-3 µm. Homocysteinylation

clusters of electrons within cells are cited as a few cases of mesoscopic

of αS1- and β-caseins results in formation of regular spheres.

biophysical phenomena. The theoretical treatment of these systems,

Homocysteinylated κ-casein forms thin unbranched fibrils about 400-

especially in a biological environment, is a hard problem, however,

800 nanometers long. In case of κ-casein amyloidogenic effect of

computational methods could provide valuable insight. Since the

homocysteinylation was confirmed by Congo red spectra. Taken

dimensions are favorable at these scales, viable experimental methods

together, data indicate that N-homocysteinylation provokes significant

and techniques could be developed to tap these interesting

changes in properties of native caseins and PrP. A comparison of

phenomena and effects. The talk concludes with an exhaustive

amyloidogenic transformation of 3 different casein types, belonging to

introduction to the major challenges in developing techniques for

the IUP protein family, shows that the efficiency of amyloidogenic

probing living systems at the mesoscopic scale (under physiological

transformation upon homocysteinylation depends on micellization

conditions). If these challenges could be successfully circumvented, a

capacity, additional disulphide bonds and other structural features.

whole new field of science could be opened, where novel theoretical ideas like possible electromagnetic channels for signal transduction and

Keywords: Homocysteinylation, Intrinsically Disordered Proteins,

inter-cellular signaling via weak IR links could be appreciated.

Aggregation, κ-caseins. Keywords:

Mesoscopic

Biophysics,

Transduction, Electromagnetic Channels.

A120

Cell

Signalling,

Signal


Abstract No.282

Keywords:

HSA,

Pd(II)

complex,

Thermodynamic

Parameters,

Circular Dichroism, Dithiocarbamate. Spectroscopic Studies of Human Serum Albumin Upon Interaction with An Anti-Tumor Pd(II) Complex Abstract No.283

Maryam Saeidifar* 1, Hassan Mansouri-Torshizi 2, Adeleh Divsalar 3, Ali.Akbar Saboury 4

4D-QSAR and Docking Study on the Pan Class I Phosphoinositide-3-Kinase (PI3K) Inhibitors:

1. Nanotechnology and Advanced Materials Department, Materials and

A Comparison to CoMFA Modeling

Energy Research Center, Karaj, IR 2. Department of Chemistry, University of Sistan and Baluchestan,

Reihaneh Safavi, Jahan B. Ghasemi*

Zahedan, IR 3. Department of Biological Sciences, Tarbiat Moallem University,

Chemistry Department, Faculty of Sciences, K. N. Toosi University of

Tehran, IR

Technology, Tehran, IR



(E-mail: [email protected]) At least one Holy Grail for many academic researchers and Protein is an important chemical substance in our life and one of the

pharmaceutical

main targets of all medicines in organism. Serum albumin, the most

therapeutically useful selective PI3K inhibitors. The class I PI3Ks is

abundant protein in the circulatory system, is one of the most

currently investigated and attracted as a promising target toward

extensively studied proteins because it can interact with many

anticancer therapies. A 4D-QSAR has been carried out on a series (42

endogenous and exogenous substances [1]. Binding of drugs to

compounds) of PI3Ks inhibitors. This approach makes use of the

plasma protein is an important pharmacological parameter, since it

molecular dynamics (MD) trajectories and topology information

frequently affects the distribution and elimination of a drug as well as

retrieved from the GROMACS package. This new methodology is based

the duration and intensity of its physiological action. The studies on

on the generation of a conformational ensemble profile, CEP, for each

this aspect can provide information of the structural features that

compound instead of only one conformation, followed by the

determine the therapeutic effectivity of drugs, and have been an

calculation intermolecular interaction energies at each grid point

interesting research field in life science, chemistry, biochemistry and

considering probes and all aligned conformations resulting from MD

clinical medicine [2]. Therefore, in this investigations, we present our

simulations. These interaction energies are independent variables or

results on the interaction studies of HSA with an antitumoral water-

descriptors employed in a QSAR analysis. We developed the method by

soluble palladium(II) complex, which possesses dithiocarbamate and

using docked multiple reference compounds as bioactive conformations

1,10-phenanthroline ligands because of modulating activity and toxicity

in alignment step for building several regression models. The

of platinum based drugs. The binding properties of this complex to

comparison of the proposed methodology to comparative molecular

Human serum albumin were studied using absorption spectroscopy

field analysis (CoMFA) formalism was performed. This methodology

and Circular dichroism techniques under physiological condition at 300

explores jointly the main features of CoMFA and 4D-QSAR models.

K and 310 K.Spectroscopic data would be used to quantify binding

Leave-N-out cross-validation (LNO), Y-randomization and application

parameters, number of binding site and the binding constants of Pd(II)

domain analysis (AD) of the obtained model were performed in order

complex to HSA and thermodynamic parameters, , molar Gibbs free

to confirm the robustness of the model in addition to analysis of the

energy of binding, , molar enthalpy of binding and, molar entropy of

independent test set. Statistical parameters of the best 4D-QSAR

binding. In addition, the experimental result indicated that this

model are (R2 = 0.941, q2LOO = 0.691, R2Pred = 0.751). Docking

complex interacted with HSA. In general, we can assert that promising

study was applied to investigate the major interactions in protein-

results obtained for the antitumor agent presented in this work make it

ligand complex with CDOCKER algorithm. Visualization of the

possible candidates for the treatment of cancer and encourage us to

descriptors of the best model helps us to interpret the model from the

design new complexes, which have better antitumor activity and

chemical point of view, supporting the applicability of this new

helpful in the development of their potential biological, pharmaceutical

approach in rational drug design. Excellent statistical parameters and

and physiological implications in the future.

the suitable predictive ability of the results explain that this model can

research

divisions

alike

has

been

to

identify

A121


help to rational design of novel PI3Ks inhibitors with preferred activities.

2. Laboratory of Microanalysis, Institute of Biophysics and Biochemistry, University of Tehran, IR (E-mail: [email protected])

Keywords:

4D-QSAR,

CoMFA,

CDOCKER,

Molecular

Dynamic

Simulation, Phosphoinositide-3-Kinase (PI3K) Inhibitors.

Gold nanoparticles (GNPs) are very attractive labels due to their special physical and chemical properties such as ease of synthesis, simplicity of conjugation and excellent biocompatibility. Because of these advantages they can be used as the carrier for a large number of

Abstract No.284

markers and bio-catalysts. In the present work, at first luminol was Hydrophilicity of Ionic Liquids Plays Important Role

attached to GNPs then the modified GNPs were chemically bound to

on Choline Oxidase Electron Transferring

antibody as a secondary antibody (Ab2-GNPs). The characteristics of chemically modified antibody were investigated by three methods

Hedayatollah Ghourchian*, Parvaneh Rahimi

including: UV-Vis spectroscopy, chemiluminescence emission and ELISA (Enzyme linked immuno-sorbance assay). The native antibody

Laboratory of Microanalysis, Institute of Biochemistry and Biophysics,

showed a UV-Vis absorbance at 521 nm wavelength.

But after


chemical modification it has a broad peak along with 10 nm red shift


compared to GNP. In second method, Ab2-GNPs were added to Ab1Ag (primary antibody (Ab1) bonded to antigen (Ag)), after washing,

Room-temperature ionic liquids (RTILs) and their related nano-

chemiluminescence

composites attracted considerable attention because of their desirable

chemiluminescence emission shows antibody preserves its function in

emission

was

recorded

in

425

nm.

High

properties and potential applications in biomolecular immobilization,

conjugation with GNP-Luminol. Preservation of antibody function has

biocatalysis, electrochemical biosensors and bioreactors. In the present

been proved by ELISA method too. This bio-composite proposed for

report, six different nano-composites contaning the same amine

diagnostic and medical usage, due to its praiseworthy detection limit of

functionalized multi-walled carbon nanotubes (NH2-MWCNTs) but

antigen in this immuno-sensors (14 pg/ml).

different room temperature ionic liquids (RTILs) were prepared. Then, the efficiency of these nano-composits as supporting materials for

Keywords: Gold Nanoparticles, Chemiluminescence, Immuno Sensor,

studying the electrochemistry and electrocatalysis of choline oxidase

Antibody Function, Antigen Detection.

(ChOx) as a model enzyme were compared. The corresponding cyclic voltammetric and amperometric data showed that the electrocatalytic activity and the electroanalytical performance of immobilized ChOx

Abstract No.286

depend on the degree of hydrophilicity of RTILs used in the applied nano-composite. The higher stability (180 days), more enzyme loading

Fine Structural Analysis of Human Serum Albumin

(6.56 M cm-2), lower detection limit (3.85 µM) and wider linear range (0.005-0.8 mM) were obtained for the most hydrophilic RTIL (1-allyl-3-

Mostafa Rezaei-Tavirani* 1, Roya Tadayon, Minoo Shahani 2

methylimidazolium bromide). 1. Shahid Beheshti University of Medical Sciences, Tehran, IR Keywords: Nanocomposite, Choline Oxidase, Electron Transfer, Functionalized Carbon Nanotubes, Ionic-Liquid, Choline.

2. Department of Base Science, Science and Research Branch, Islamic Azad University, Tehran, IR (E-mail: [email protected]) Human serum albumin is the most abounding protein in human blood

Abstract No.285

plasma. It plays essential roles such as maintaining pH level and Chemical Modification of Antibody Using

osmotic pressure in blood. This protein possesses a particular

Gold Nanoparticles Bearing Luminol

adequacy to bind to a great number of various endogenous and exogenous compounds. This article outlines different features of HAS

1

Soheila Sabouri , Hedayatollah Ghourchian*

2

and its multifunction eligibility. These studies can be obtained by pH metery, ligand binding, UV spectroscopy, fluorescence spectroscopy


A122

and CD techniques. The results compared and discussed based on data


achieved by these methods. Statistical analysis has been revealed at

energy of the system with respect to the initial state (∆U) along the

least six fine individual structures for HSA. Each structures present

minimum energy reaction path (MERP) is also reported with the 3D

individual and exclusive function. It can be concluded that, albumin

structures of relevant intermediates. The results are consistent with

unique features and functions are due to its switching capability to

the available experimental data and provide new insight into the

different possible fine structures with the lowest energy exchange.

detailed mechanism of this important reaction.

Keywords: Human Serum Albumin, Fine Structures, Structural And

Keywords: Quantum Mechanic, Molecular Mechanical, Thioredoxin,

Functional Alteration.

Glutathione Reductase.

Abstract No.287

Abstract No.288

Mixed Quantum Mechanical/ Molecular Mechanical (QM/MM)

Effect of Glycine, as Chemical Chaperone, on Catalase

Study of the S-nitrosylation Reaction 4-phenyl-3-

Glycation by Glucose

Furoxancarbonitrile as Inhibition of Thioredoxin Glutathione

Fereshteh Bahmani* 1, S. Zahra Bathaie 1, S. Javid Aldavood 2,

Reductase (TGR)

Arezou Ghahghaei 3 1

2

Fereshteh Shiri , Jahan B. Ghasemi* , Paolo Tosco

3

1. Department of Clinical Biochemistry, Faculty of Medical Science, 1. Faculty of Chemistry, Razi University, Kermanshah, IR


2. Department of Chemistry, Faculty of Sciences, K. N. Toosi University

2. Department of Clinical Sciences, Faculty of Veterinary Medicine,

of Technology, Tehran, IR


3. Department of Drug Science and Technology, University of Turin,

3. Department of Biology, Faculty of Science, University of Sistan and

Combined

Via Pietro Giuria 9, 10125 Turin, IT

Baluchestan, Zahedan, IR



quantum

mechanics/molecular

mechanics

(QM/MM)

Oxidative mechanisms are thought to have a major role in several

methods allow computations on chemical events in large molecular

biological phenomena, including cataract formation and diabetic

systems. We present a theoretical study of a mechanism for the S-

complications. Glycation of catalase, as a powerful antioxidant enzyme,

nitrosylation Reaction4-phenyl-3-furoxancarbonitrile or furoxan belongs

by glucose alters its structure and function and increases the risk of

to an oxadiazole-2-oxide class whose therapeutic effects are largely

cataract formation in diabetic patients. Glycine is a chemical

due to their inhibition of thioredoxin glutathione reductase (TGR)for

chaperones that inhibit protein glycation and stabilize them against

thecontrol of schistosomiasis bases in QM/MM calculations at the DFT

thermal and chemical denaturation. Therefore, we investigated the

RB3LYP/6-31G+(d)//SCC-DFTB AMBER level. The activity of the

effects of this chemical chaperone on catalase glycation.Catalase

oxadiazole 2-oxides was associated with a donation of nitric oxide. We

solution, pH 7.4, was incubated separately with glucose in the

first attempted to dock inhibitor in the active site of enzyme available

presence or absence of glycine at 37ºC in a shaker incubator. The

in the Protein Data Bank, (PDB codes: 3H4K), to see how intact

aliquots were collected every week and stored at -80ºC. Then, they

inhibitor would bind. Since our interest was focused on the region were

were analyzed by fluorescence spectroscopy, circular dichroism

reaction should take place, all molecules that might potentially be

spectroscopy (CD) and polyacrylamide gel electrophoresis (PAGE). The

involved in the mechanism of nucleophilic attack, namely furoxan,

activity of catalase in each sample was also determined.Structure and

residues Cys159, and nearby water molecules, were treated by QM,

activity of catalase were changed due to the glycation with glucose.

while the rest of the protein, as well as the bulk water, were treated by

Glycation caused an increase in the fluorescence emission and

traditional molecular mechanics. As soon as a preliminary minimization

electrophoretic mobility and a decrease in the alpha helix content and

of complex was carried out on TGR using the hybrid QM/MM potential,

catalase

migration of the Cys159 to the carbon 3 furoxan was observed. We

fluorescence emission, alpha helix content, electrophoretic mobility and

were quite surprised by this outcome, while the formation of the

activity of catalase were closed to the normal values.Our results

tetrahedral intermediate might be a reasonable explanation for location

indicated a decrease in the catalase glycation by glucose in the

activity.

Glycine

inhibited

this

phenomenon

and

the

of the initial nucleophilic attack shows this mechanism: The potential

A123


presence of glycine. It implies the usefulness of this\e chemical

Abstract No.290

chaperone in reduction of diabetic complications such as cataract. Biotechnological Applications of Peroxidases Keywords: Catalase, Chemical Chaperone, Glycation, Glycine.

Khodadad Nazari* 1, Ali Akbar Moosavi-Movahedi 2 1. Chemical Sciences and Technology Division, Research Institute of

Abstract No.289

Pe-troleum Industry, 18745/4163, Tehran, IR Thermodynamic Aspects of Disulfide Bridge Introduction and

2. Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IR

Bioluminescence Color Shift in Firefly Luciferase


Saman Hosseinkhani* 1, Mahboobeh Nazari 1, Leila Hassani 2 Peroxidase enzymes that may find ubiquitously in animals, plants and 1. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat

microorganisms, are able to oxidize a wide range of reductants in the


expense of peroxides as acceptors[1,2]. Most part of our knowledge on

2. Department of Biological sciences, Institute for Advanced Studies in

the structure–function relationships and catalytic mechanisms of

Basic sciences (IASBS), Zanjan, IR

peroxidases

is

indebted

on

horseradish

peroxidase.In

classical


peroxidases, ferric protoporphyrin IX is the prosthetic group and imidazole the fifth iron ligand. The characteristic activity of peroxidases

Relationship between stability, color shift and position of flexible loop

is two electron oxidation, which proceeds through formation of

in firefly luciferase is investigated. Bioluminescence reaction, which

compounds I and II. Compound I is reduced back to the ferric resting

uses

an

state either by a two sequential one-electron transfer processes from

electronically excited oxyluciferin, is carried out by the luciferase and

peroxidase to substrates or by two-electron oxidation process

emit visible light. Multi-color bioluminescence is developed using

associated with the ferryl oxygen transfer to substrates[3]. The

introduction of single/double disulfide bridges in firefly luciferase

radicals produced in the reaction generally evolve nonenzymatically to

through structural stabilization and displacement of functional loops.

nonradical products by pathways characteristic of each substrate

The bioluminescence color of firefly luciferases is determined by the

(coupling, dismutation etc.).Chloroperoxidase (CPO) is unique among

luciferase structure and assay conditions. Single and double disulfide

the peroxidases because it contains a cysteinic thiolate as the fifth axial

bridges are introduced into firefly luciferase to make separate mutant

ligand of the heme instead of the imidazole ligand. For this reason,

enzymes.

luciferin,

Mg2+-ATP and

molecular oxygen

to yield

By introduction of disulfide bridges using site-directed

many of its spectroscopic and chemical properties are similar to those

mutagenesis in Photinus pyralis luciferase the color of emitted light

of cytochrome P-450. CPO is unusually versatile: it catalyzes not only

was changed to red or retained. Multicolor bioluminescence is

the reactions typical of peroxidases but also those of catalases and

accompanied with displacement of critical loops in red-emitter

monooxygenases,

and it

is

also almost

unique in

catalyzing

luciferases without significant changes in green emitter mutants.

halogenation reactions in the presence of halide ions and H2O2.

Thermodynamic analysis revealed that among mutants, L306C-L309C

Peroxidase-catalyzed reactions exist in four categories[4]:1. Oxidative

mutant with a single disulphide bridge shows a remarkable stability

dehydrogenation 2.Oxidative halogenations 3. H2O2 dismutation 4.

against urea denaturation and also considerable increase in kinetic

Oxygen-transfer

stability with changes in emission spectra towards red.

oxidation, Epoxidation, Indole oxidation, Heteroatom oxidation (Sulfur

reactions:

Benzylic/allylic

hydroxylation,

Alcohol

oxidation, Nitrogen oxidation)Present work reviews and discusses Keywords: Bioluminescence, Disulfide Bridges, Luciferase, Red-

recent biotechnological developments and applications of peroxidase

Emitter, Site-Directed Mutagenesis.

family including homogeneous, heterogeneous, artificial peroxidases and miniperoxidases. Keywords: Peroxidase, Chloroperoxidase, Application, Reaction.

A124


simulation and homology modeling that used in the structure

Abstract No.291

determination so that some of PDB codes in protein data bank were Lys Therapy of Diabetes: from in vitro Studies to the Animal

exclusively obtained by MD. On the other hand X-ray crystallography

Models of Diabetes and Type 2 Diabetic Patients

only obtains the crystal structure of macromolecules but not solution. NMR spectroscopy investigates both liquid and crystal structures while

S. Zahra Bathaie*

CD and IR determine secondary structure in solution phase. The first two methods are precise and rigorous but very expensive. On the

Department of Clinical Biochemistry, Faculty of Medical Sciences,

other hand MD is a powerful method in determination of tertiary


structure of macromolecules specially in homologues proteins. MD


methods use the classical and statistical mechanics theorem for movement and interaction of molecules. So it is an approximation

Hyperglycemia is one of the most important reasons of diabetic

method that dose not consider electron interaction in its calculations. It

complications. It causes the biomacromolecules, especially protein

makes atoms to move in different direction based on Newtonian laws

glycation which in order result in protein conformational change. Here

subsequently thermodynamic parameters. In this study, I report some

the effect of glycation on the structure and function of several

applications of molecular dynamics in biology. Structural parameters

important

plasma,

such as solvent accessible surface area, hydrogen bond, CD222nm,

extracellular medium, cytosol and nuclei is presented. Then, the effect

radius of gyration and radial distribution function were obtained for

of Lys, as a chemical chaperone from amino acid family, on the non-

enzyme by molecular dynamics and compared with experimental data.

proteins

from

various

comportments

e.g.

enzymatic glycation of many proteins and its inhibitory effect on this process that was investigated in our Lab. will be discussed. In addition,

Keywords: Molecular

our in vivo results showed that Lys administration in the animal model

Accessible Surface Area, Hydrogen Bond.

Dynamics

Simulation,

Tertiary

Structure,

of diabetes of both types 1 and 2, as well as the type 2 diabetic patients result in the decrease in serum glucose, increase in insulin secretion and decrease insulin resistance, increase serum antioxidant

Abstract No.293

capacity, improve the lipid profile and HDL functionality, induce HSP70 production, reduce fibrin activity due to correction of its folding, stop

New Strategies in Structure-function Relationship Study of

the progression of cataract, etc. In conclusion, the obtained data by

Peptides

our team in the in vitro and in vivo studies indicate the beneficial effects of Lys therapy on reduction of diabetic complications thus it is

Bahram Hemmateenejad*

suggested as a complement therapy for diabetes. Chemistry Department, Shiraz University, Shiraz, IR Keywords: Diabetes, Animal Model, Hyperglycemia, Antioxidant.

(E-mail: [email protected]) Peptides play significant roles in biological world especially in human life. There are a great number of peptides and proteins which are used

Abstract No.292

as therapeutics and more are under development as pharmaceutical Molecular Dynamics Simulation on the Protein Structure

targets. However, design and prediction of their activity remain one the most challenging area in life sciences due to large amount of

Davood Ajloo*

arrangement

possibilities.

Quantitative

sequence-activity

model

(QSAM), employs quantitative structure-activity relationship (QSAR) 1. School of chemistry, Damghan University, Damghan, IR

strategies to quantify biosequence-activity/function relationship for the


peptides, proteins and nucleic acids, becomes an attractive and active


area in peptide researches. Therefore, a lot of efforts were done in the past to model the relation between the peptide structure/sequence

The protein structure changes in the presence of different denaturants

with its biological activity. On the basis of QSAR concept, functions and

such as temperature and chemicals. These variations can be followed

structures of peptides or proteins are resolved by the information

by different techniques such as X-ray, NMR, CD, IR and so on. There

enclosed in the amino acid arrangements. In this methodology, a set of

are some complementary methods same as molecular dynamics (MD)

A125


descriptors is calculated for each amino acid and they are put together

substrates and TMB, L-Dopa or DOPA as a reductant substrates.

to produce a descriptor data matrix for a set of peptides.

Results showed that the non-specific peroxidase activity of the ‘‘heme-

Very recently, in our research group we have been involved in the

ferritin” system can oxidize the neurotransmitters with high catalytic

definition of some new amino acid indices (AAI) for use in QSAM study

efficiency. According to the literature, the concentration of ferritin, free

of peptides. We employed the concept of atom in molecule and used

heme, and peroxide species increase in neurodegenerative diseases

the quantum topological molecular similarity (QTMS) descriptors to

such as AD. There is this possibility that the peroxidase activity of

suggest some new AAIs. In addition, we employed the Moreau and

‘‘heme-ferritin” complex play a significant role in development of

Broto autocorrelation function on the QTMS descriptors of amino acids

oxidative damage within involved cells. We will discuss the importance

to extend the QTMS-based AA indices and generate some other novel

of our observations.

indices. In another work, to increase the quality of the QSAR models reported for peptides, we proposed here the application of segmented

Keywords: AD, Peroxidase Activity, Ferritin, Neurodegenerative

principal component regression (SPCR) method to define more relevant

Diseases.

sources of AA indices. Finally, using of multi-way data analysis methods in QSAM study of peptides will be explained. The suggested methods were validated by analysis of some different peptide data sets

Abstract No.295

of relevant biological activities. Interaction of a Novel Photochromic Molecule with Human Keywords: Amino acid indices, Peptide, Biological activity, Structure-

Serum Albumin Studied by UV-vis Absorption, Fluorescence

function.

Spectroscopy, and Docking Calculation

Fereshteh Shaheri 1, Davood Ajloo* 1,3, Hamzeh Kiyani 1, Maryam Ghadamghahi 1, Taghi Lashkarbolooki 2,3

Abstract No.294

1. Department of chemistry, Damghan University, Damghan, IR

A Possible Role of Peroxidase Activity of ‘‘heme-ferritin”

2. Department of Biology, Damghan University, Damghan, IR

Complex in Development of Neurodegenerative Diseases


Morteza Jafari* 1,3, Reza Khodarahmi 2, Samira Ranjbar 3, 3

Mohammad Reza Ashrafi* , Seyyed Mohsen Asghari


4

Photochroism is the phenomena that of reversible transformation 1. Dept. of Biology, Faculty of Science, Guilan University, Guilan, IR

between two forms (open and close form) that have different spectra

2. Medical Biology Research Center, Departments of Pharmacognosy and

by photo-irradiation. In this work, the interaction of a new

Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical

photochromic molecule, 2,2-biphenyl-4-yl-2-methyl-6(4-nitrophenyl)-4-


phenyl-1,3-diazobicyclo[3.1.0] hex-3-en, with human serum albumin


(HSA) has been investigated by UV-vis absorption, fluorescence


spectroscopy and docking calculations. 0.1 µM HSA was used in the

4. Dept. of Biology, Faculty of Sciences, Guilan University, Rasht, IR

presence of 0 to 1.47×10-5 M photochromic molecule. The interaction


of photochromic molecule with HSA causes fluorescence quenching of HSA. The addition of photochrome to a solution of HSA induces

Ferritin is a ubiquitous protein for the sequestration, storage and

quenching of the protein fluorescence and binding constant of

release of iron in animal and plant cells. It has been previously shown

interaction was also obtained about 7. 5 µM-1. In other investigation

that each ferritin molecule can bind to 15 - 17 heme moieties. Heme

the photochromic molecule absorbance was studied by a UV-Vis

and sometimes heme-protein complexes possess peroxidase activities

spectrophotometer model Perkin Elmer. The photochromic reaction

which cause irreversible damages in cells. In the present study, we

was

reported the peroxidase activity of “heme-ferritin” complex using

spectrophotometer. Absorption spectra of photochromic molecule and

several oxidizable substrates. First, the ferritin was purified from liver

HSA were obtained as the time of irradiation increase from 0 to 70 s.

homogenate of sheep by ammonium sulphate precipitation and DEAE

Absorbance intensity was increased due to the close form of molecule

ion-exchange chromatography. The peroxidase activity of ‘‘heme-

that turn in to the open form. Rate constant of reaction was obtained

ferritin” complex was then measured by using H2O2 or t-BHP as oxidant

from absorption spectra about 0.026 s-1. In order to estimate the

A126

studied

upon

variation

of

time

irradiation

by

UV-vis


affinity and binding energy of photochromic molecule to HSA, Autodock 3.0.5 software was used. The negative value of docking energy has revealed interaction of protein with this molecule. Keywords: Photochromism, Human Serum Albumin, Rate constant.

Abstract No.296 Electrochemical Characterization of Cytochrome C by Using the Electrode Modified with Cadmium Selenium Nanoparticles

Saeid Rezaei-Zarchi 1, Saber Imani* 2, Ali Mohammad Zand 3, Yunes Panahi 2, Amir Nejad Moghaddam 2, Eisa Tahmasbpour Marzon 2 1. Department of Biology, Payame Noor University, Yazd, IR 2. Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR 3. Department of Biology, Basic Science Faculty, IHU, Tehran, IR (E-mail: [email protected]) Bare surface of the electrode in electrochemical studies of proteins are not suitable. In this case, leads to a decrease in the rate of electron transfer between electrode and protein and protein irreversible adsorption on the electrode surface which is associated with conformational changes and loss of protein activity. Therefore, you must provide the necessary groups on the electrode surface for active communication with the macromolecules. Molecules could provide the group called facilitator. In this paper, was diagnosed with cytochrome c with modified glass carbon electrode surface by cadmium selenium nanoparticles. This detection can be used in Biosensors designed for measurement of hydrogen peroxide. X-ray Diffraction (XRD) results showed size nanoparticles synthesized with electrochemical method are 57 nm. Alpha Absorption

spectra

revealed

the

size

limit.

Advantage

of

electrochemical methods for synthesis of nanoparticles of cadmium selenium is an easy, cheap and low cost. Biosensors with cytochrome c stabilized on this electrode shows high sensitivity and linear range from 15 to 700 micro M for determination of hydrogen peroxide. Keywords:

Cytochrome

C,

Cadmium

Selenium

Nanoparticles,

Electrochemical Characterization.

A127