Iranian

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Many organisms produce lactic acid by fermentation, but most industrially important strains are from the genus Lactobacillus and Rhizopus oryzae. L(+)-Lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. In this research, six strains of Lactobacillus and four strains of R. oryzae (known as high producer) were examined for optical isomers of lactic acid. The production of lactic acid was improved and lactic acid produced in submerged media on rotary shaker incubator. The optical isomers of lactic acid were examined by L(+) and D(-) lactate dehydrogenase kit. All the R. oryzae strains tested produced only L(+) isomer of lactic acid. The highest fungal and bacterial producer strains were R. oryzae PTCC 5263, Lactobacillus plantarum PTCC 1058, L. Bulgaricus PTCC 1332 and L. delbruekii subsp delbruekii PTCC 1333. Lactobacilli strains produced combination of both optical isomers of lactic acid. Among them, L. casei subsp. Casei produced the low amount of D(-)-lactic acid (2%). The optimum concentration of glucose for lactic acid production by R. oryzae and Lactobacillus strains were 180 g/l and 80–120 g/l, respectively.

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Penicillin acylase (EC 3.5.1.11) has been a target of study for a long time because of its pivotal role in the deacylation of the penicillin into the 6- aminopenicillanic acid (6-APA) and the side-chain organic acids. This property of penicillin acylase has been exploited commercially for large scale production of 6-APA, which is the key intermediate in the manufacture of semi-synthetic penicillins. Due to the worldwide demand for semi-synthetic penicillins, production of 6-APA has been increased up to 7000 tons in recent years. In this study, Sixty-five strains of E. coli were investigated for penicillin acylase activity using fluorescamine method. The 6-aminopenicillanic acid formed in the reaction mixture was developed on thin layer chromatography. One-minute beta-lactamase test was carried out to follow any trace of penicillinase activity. Only one sample designated as E.coli PPA78 was found to be penicillin acylase producer. The optimal pH and temperature of penicillin acylase activity of the whole cells were determined to be 8.0 and 57° C, respectively. Km value and activation energy of the enzymatic hydrolysis reaction of penicillin G by intracellular enzyme were estimated as 0.004 mmol and 6.2 Kcal/mol, respectively.

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Human diploid fibroblast cells produce a spectrum of necessary growth factors and extracellular matrix (ECM) components essential for growth and proliferation of a variety of other cell types. In this study, the effect of five human embryonic fibroblast cell lines, isolated from liver, lung, skin and foreskin tissues, was investigated. A coculture system analyse was employed to cloning efficiency (CE) and DNA synthesis of a human Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) in long- and short-term cultures. The fibroblast cells were used as feeder layer after treatment with mitomycin C. Optimal density of the feeder cells induced 10 to 43 times higher CE than cultures supplemented with conditioned media (CM) or cultures without a feeder layer. The stimulatory effect of the feeder cells was partly associated to their tissue origin, with the lung and liver fibroblasts being the most and least effective feeder cells, respectively. Short-term cultures of LCL cells with feeder cells or their CM resulted in a marginal increase in DNA synthesis and proliferation as evidenced by the index of ³H-thymidine incorporation. Our results demonstrated supportive effects of feeder cells on the LCL growth, which can not be replaced by their CM. These supportive effects were partly associated with cell density and tissue origin of the feeder cells

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Cladribine, an analogue of deoxyadenosine, is highly toxic for both non-dividing and proliferating cells and has shown activity in the treatment of several malignancies. Therefore, the aim of the present study is to investigate the cytotoxicity effect of cladribine (2-CdA) on the breast cancer cell line, MCF-7 (estrogen receptor positive, ER+). MTT assay, annexin V-Fluorescein/PI and Hoechst 33258 staining were used to detect cytotoxicity and cell apoptosis. The activation of caspase-3 and -9 was assayed using caspase activation assay kits. Gel electrophoresis was performed to detect DNA fragmentation. Treatment of MCF-7 cells with different concentrations of 2-CdA resulted in a significant increase in the cell death. Annexin V-Fluorescein/PI and Hoechst 33258 staining revealed that the cell death was mainly an apoptotic type. A significant (p<0.05) increase in the activity of caspase-9 was observed but Caspase-3 activity was unchanged and DNA laddering profile was not obtained. Pre-treatment of the cells with kinase inhibitor, 5¢-amino-5¢-deoxyadenosine inhibited the cytotoxicity effect of cladribine. In conclusion, this study has shown that high dose of cladribine (higher than 25 μM) has an apoptotic effect on MCF-7 cells and that its intracellular phosphorylation is necessary

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b-cyclodextrin glucosyl transferase (b-CGTase) hydrolyses starch to produce b-cyclodextrin by transglycosylation (cyclization) activity. The conventional method for detection of b-CGTase activity is based on detecting starch hydrolysis by iodine staining. This method reveals all amylolytic enzymes, but can not discriminate them. In the present study, we introduce a new method for specific detection of b-CGTase activity and its specific product i.e. b-cyclodextrin by polyacrylamide gel. In this method, solution containing b-CGTase is subjected to electrophoresis on 10% polyacrylamide gel. Then, the gel is covered with an indicator gel containing phenolphthalein, soluble starch, and agar. After a short incubation, sodium carbonate solution is added and the positive result is indicated by the emergence of a colorless band in the red context of the indicator gel. Since the production of b-cyclodextrin via cyclization is unique to b-CGTase, the emergence of clear bands is indicative of b-CGTase presence. However, in conventional starch-iodine method, all amylolytic enzymes including b-CGTase give positive results. Therefore, for detection of b-CGTase, the phenolphthalein indicator gel method, compared to the starch-iodine method, is more specific

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 The efficacious effects of pulsed electromagnetic field (PEMF) under the certain field parameters like frequency and the field intensity have been reported for various tissue and molecules. Since collagen is found abundantly in most tissue structures, this research was designed to further investigate the effects of extremely low frequency (ELF) PEMF on the synthesis of the epidermal collagen. To do the task, six groups of animals each consisting of eight mature male rats were selected randomly as one group for the control and five for the test. The field was generated by using a parallel set of Helmholtz coil. The first set of experiments was carried out at the peak intensity of 2 mT (milli Tesla) for different frequencies of 25, 50 and 100 Hz. Since the most effective frequency turned out to be 25 Hz, another set of experiment was conducted using this frequency and two different field intensities of 1 and 4 mT. The field was applied for 2.5 h/day lasting for 8 days, keeping the same procedure for the control group except for the field turned off. On the ninth day, the rats were sacrificed and the skin samples from the dorsal region were taken for biochemical assessment of collagen by measuring hydroxyproline content using Stegeman-Stalder method and histological assessment. The data indicated that pulsed electromagnetic field of 2 mT at 25 Hz increased the collagen synthesis (P<0.05). The other intensities and frequency setting did not have much distinguishable effect, however, at the frequency of 25 Hz and 4 mT, the field effect on the collagen increase was also noticeable. It was concluded that applying the field parameters of 25 Hz and 2 mT peak intensity for 2.5 h/day during eight days rendered a significant increase in collagen synthesis in rat skin. Histological observations were consistent with the biochemical findings.  

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The aim of this study was to compare invasive and non-invasive strains of Shigella flexneri isolated from Tehran by a 120 kDa protein band by SDS-PAGE, electron microscopy of cell culture and Congo red dye methods. Methods: S. flexneri strains were isolated by standard bacterial methods from fecal specimens of children attending to the 3 children’s hospitals. Phenotype analysis for screening virulent of strains of S. flexneri was done on a plate of tryptic soy agar contained 0.003% Congo red dye. Whole membrane protein preparations were used to examine the protein profiles of the inner and outer membrane of these Gram-negative bacteria. The protein mixture was electrophoresed through a polyacrylamide gel. The gel was stained with Coomassie brilliant blue R250 and destained with ethanol and acetic acid. HeLa cell culture was done by two-step preparations: one for light microscopy and the other for electron microscopy. Results: Some of S. flexneri (46%) were Congo red positive colonies. S. flexneri with negative Congo red phenotype could not enter the HeLa cell culture. A 120 kDa protein band was found in 46% of these bacteria which could enter into HeLa cell culture. Pseudopod structures which facilitate bacterial cell-to-cell spread were readily identified by electron microscopy. Discussion: Since the existence of 120-kDa protein band was corresponded to enter of S. flexneri into the HeLa cell culture and correlated with Congo red dye positive, for identification of invasive and non-invasive S. flexneri strains, the use of a 120-kDa protein band by SDS-PAGE or a simple, rapid and very cheap Congo red dye method is recommended. Because, there are some deaths due to Shigella sp. in our country, notification on the isolation of these bacteria in both children hospitals laboratories and private clinical laboratories is important.

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The human leukocyte antigen G (HLA-G) molecule exhibits limited tissue distribution, low polymorphism and alternative splicings that generate seven HLA-G isoforms. HLA-G exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells that may favor their escape from anti-tumor immune responses. This study it is an effort to clarify the presence of HLA-G in B-cell chronic lymphocytic leukemia (B-CLL) patients. Methods: HLA-G mRNA expression was studied in a pilot study in circulating B-CLL and also healthy controls by reverse transcription (RT)-PCR using a set of pan-HLA-gamma primers. Results: RT-PCR was performed on B-cells from 74 B-CLL patients and 12 healthy controls. The data showed HLA-G gene expression in 20% of the B-CLL patients. No expression of HLA-G could be detected in the healthy control group. Conclusion: These data suggest that HLA-G is expressed at the gene level in B cells from B-CLL patients but not in B cells from healthy controls. Further study is required to clarify the role of HLA-gamma as a regulatory factor that could affect immune response in B-CLL patients.

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Objectives: It was aimed to investigate the effects of different doses of follicle stimulating hormone (FSH) and activin A on the growth and maturation of preantral mouse follicles during the in vitro culture. Methods: Preantral follicles (90-100 µm in diameter) were harvested from 6-8 week-old Syrian mice and cultured in TCM199 culture medium for 6 days to see the effect of FSH and Activin A. Activin A concentrations in the range of 10-200 ng/ml were used, while 10, 50, 100, 150 and 200 mIU/ml FSH were used in the experiment. Results: Activin A concentration of 100 ng/ml resulted in a significant increase in follicle diameter (170 µm) with the survival rate of 73% as compared to the control (100 µm and 25%, P<0.05). The number of oocytes matured and the percentage of germinal vesicle breakdown (GVBD) was 61 and 70%, respectively as compared to the control (20 and 29%, P<0.05). Follicle diameter (190 µm) and survival rate (85%) increased significantly in the presence of 100 mIU/ml of FSH as compared to the control (P<0.05). But, the administration of activin A+FSH increased the effect of both factors on follicular diameter (205 µm as compared to 100 µm in control, P<0.01). Follicle survival, oocyte maturation and GVBD rates were 91, 81 and 89%, respectively (P<0.01). Conclusion: These results have suggested that exposure to FSH and activin A before the formation of antral cavity had positive effect on follicle survival and oocyte robustness.

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Background: Vancomycin (glycopeptide)-resistant enterococci (VRE or GRE) can cause serious problems for hospitalized patients due to the limited options for treatment of VRE infections. As infection with VRE increases in hospitals, further knowledge about vancomycin resistant genes is needed. Methods: Isolates of Enterococcus spp. were collected from hospitalized patients in Tehran (Iran) during 2006. Detailed molecular analysis was performed for vancomycin resistance genotype and vanHAX using conventional PCR and PCRRFLP (restriction fragment length polymorphism), respectively. Results: out of 830 enterococci spp., 48 VRE isolates (5.8%) were obtained. All of VRE isolates carried vanA gene. DdeI digestion of vanHAX element showed the presence of point mutation at 8234 position. Conclusion: This study indicates that vanA is a predominant genotype in Iranian isolates. In addition, PCR-RFLP analysis revealed the presence of two types of vanHAX element in vanA harboring transposons.

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Background: Sex steroids and their receptors exist in hippocampus and affect spatial learning and memory. This study was designed to measure testosterone level of CA1 and to assess the effect of spatial learning on its amount in left and right hippocampus of adult male rats. Methods: Sixteen rats were divided into two intact and castrated groups, and then trained in Morris water maze (MWM). Another 40 animals were divided into four groups and their right or left hippocampus cannulated. Half of the animals in each group were castrated simultaneously. All the animals were trained in MWM. Microdialysis was performed and steroid contents of hippocampal dialysate were analyzed through HPLC/ultraviolet detection device method. Results: Results showed no significant differences between control and castrated animals in spatial learning after four days of training. Gonadectomy did not change testosterone level in CA1 region of hippocampus. Spatial learning decreased testosterone levels in CA1 region of hippocampus in right hippocampus of the non-castrated group. Significant differences were indicated in testosterone level between left and right hippocampus, in favor of left side in all groups. Conclusion: Castration does not affect learning. Testosterone, as a neuromodulator, exists in CA1 region of hippocampus and training can decrease its level only in right hippocampus significantly. Lesser testosterone content of right hippocampus may show the conversion of it to other metabolites.

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Background: Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds. Methods: In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker. Results: The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells (ASC) in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis. Conclusion: These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo.

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Background: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA). Methods: Isolated pre-antral follicles (140–150 µm in diameter) were divided into vitrified–warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. Results: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. Conclusion: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and non-vitrified samples through increasing follicular TAC level and decreasing ROS levels.

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Background: Microglial cells act as the sentinel of the central nervous system .They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide (NO) and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation. Methods: Following the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain (5, 10, 20, and 30 µg/ml) on the levels of NO, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-&kappaB) in microglia treated with lipopolysaccharide (LPS) (1 µg/ml). Data were analyzed using Student's t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain. Results: Results showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS (1 µg/ml) treatment in a dose-dependent manner. Bromelain (30 µg/ml) also significantly reduced the expression of iNOS at mRNA level and NF-&kappaB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death. Conclusion: Bromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases.

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Background: This study was conducted to reveal that whether i.v. injection of oleuropein, the most potent polyphenolic antioxidant in olive leaf, has any effect on the magnitude of reperfusion arrhythmia in anesthetized rats or not. Methods: Eighty male Wistar rats were divided into 8 groups of 10 each: groups 1 and 5 were assigned as the prophylactic and treatment control groups, groups 2 and 6 as the prophylactic and treatment groups with lidocaine (10 mg/kg), groups 3 and 4 as the prophylactic groups with 10 and 50 mg/kg oleuropein (i.v.), and groups 7 and 8 as the treatment groups with 10 and 50 mg/kg oleuropein (i.v.), respectively. Reperfusion injury was induced by 5-min regional ischemia and 15-min reperfusion of left anterior descending coronary artery. Heart rate, blood pressure, and electrocardiogram were monitored throughout the procedure. Results: blood pressure was significantly decreased by infusion of 50 mg/kg oleuropein in groups 4 and 8, but unlike the lidocaine as a standard anti-arrhythmic drug in groups 2 and 5 had not significant effect on heart rate. The onset of arrhythmia in groups received oleuropein (groups 3, 4, 7, and 8) was significantly delayed. The mortality rate due to irreversible ventricular fibrillation was also significantly reduced in groups 3, 4, 7, and 8. The effect of lidocaine in groups 2 and 5 was more potent than that in oleuropein group. Conclusion: These findings indicate that i.v. injection of oleuropein possibly through its antioxidant activity reduces the magnitude of reperfusion-induced arrhythmia.

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Background: Progressive encephalopathy with or without lipodystrophy is a rare autosomal recessive childhood-onset seipin-associated neurodegenerative syndrome, leading to developmental regression of motor and cognitive skills. In this study, we introduce a patient with developmental regression and autism. The causative mutation was found by exome sequencing. Methods: The proband showed a generalized hypertonia and regression of all developmental milestones. Based on the advantages of next-generation sequencing (NGS), whole exome sequencing (WES) was requested. The functional significance of variants was evaluated by NGS-specific prediction servers. Sanger sequencing was used for segregation analysis in the family. Results: There was no specific sign in the clinical and paraclinical investigations of the patient to establish a conclusive
clinical diagnosis. WES detected a known homozygous nonsense mutation in BSCL2 (NM_001122955.3:c. 985C>T; p.Arg329*). The variant is segregating in the pedigree with an autosomal recessive pattern. Conclusion: Exome sequencing is a robust method for identifying the candidate gene variants in Mendelian traits. 

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Background: Ghrelin is a peptide with attenuating effect on inflammatory pain. Both anti- and pro-inflammatory mediators have a role in the nociception and development of pain and hyperalgesia. IL-10 and TGF-β are anti-inflammatory cytokines and inhibit the expression of pro-inflammatory cytokines related to peripheral and central inflammatory pain. In this study, the effects of i.p. injection of ghrelin on the early and the late phases of pain, as well as serum levels of IL-10 and TGF-β, as anti-inflammatory cytokines, were investigated in formalin-induced pain in male rats. Methods: Adult male Wistar rats (n=48) were randomly divided into six groups: control, formalin+saline, ghrelin (40, 80, and 160 μg/kg), and morphine. Ghrelin was administered i.p. 30 min before inducing pain by formalin. Pain induced by intraplantar (i.pl.) injection of 50 µl formalin 5%, and pain behavior was studied for 60 min. Serum IL-10 and TGF-β levels were assessed by ELISA method. Results: The findings of the present study showed that ghrelin with high doses (80 and 160 μg/kg) significantly reduced pain intensity in both the early and the late phases of pain. The serum levels of cytokines, IL-10, and TGF-β1 showed a significant elevation with ghrelin at dose of 160 μg/kg. Conclusion: Ghrelin is effective in reducing the intensity of both the early and the late phases of inflammatory pain. It seems that ghrelin exerts its analgesic effects in part by increasing the serum levels of anti-inflammatory cytokines.

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Background: Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates (ADCs). Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates. Methods: In the present study, we investigated the effect of various dithiothreitol (DTT) concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively. Results: DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively. Conclusion: Optimized site-specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs.

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Background: Non-structural protein 4 (NSP4) is a critical protein for rotavirus (RV) replication and assembly. This protein has multiple domains and motifs that predispose its function and activity. NSP4 has a sequence divergence in human and animal RVs. Recently, 14 genotypes (E1-E14) of NSP4 have been identified, and E1 and E2 have been shown to be the most common genotypes in human. Methods: The gene and protein sequence of NSP4 in RV-positive samples were inspected with the aim of NSP4 genotyping and variation analysis in viroporin and other domains. P and G typings of RV samples were carried out by WHO primers using a semi-multiplex PCR method. Non-typeable RV samples were amplified by conserved primers and sequenced. Results: In viroporin and enterotoxin, conserved sequence was detected, and amino acids substitution with the same biochemical properties was found. Conclusion: Association of NSP4 genotype with P or G genotyping G1/G9 correlates with E1 genogroups. In electrophoretyping of RV, E2 genotype had a short pattern when compared to E1.

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Background: Herpes simplex virus type 2 (HSV-2) is a common infection in human immunodeficiency virus (HIV) patients and may accelerate HIV progression by rising HIV viral load and decreasing CD4 count. However, the available data regarding the influence of HSV-2 seropositivity on HIV progression in HIV individuals are inconclusive. Therefore, we aimed to determine HSV-2 seroprevalence in naïve HIV patients and normal controls and also investigate the relation of HIV viral load and CD4 count with HSV-2 seropositivity. Subsequently, we investigated the association of HSV-2 serostatus with changing in CD4 count and HIV viral load in our subjects, after one year follow-up. Methods: In this study, 116 naïve HIV patients and 85 healthy controls from Tehran, Iran were enrolled.  HSV-2 IgG antibody was detected by ELISA. CD4 count was determined by flowcytometry, and serum HIV RNA copy numbers were determined using real-time PCR. Results: The prevalence of HSV-2 IgG was 18.1% in naïve HIV patients and 0% in the control group (P = 0.000). HSV-2 seroconversion was observed in 2.43% of HIV patients after one year. There was no significant difference regarding HSV-2 serostatus with CD4 count and HIV RNA viral load in our study cohort at baseline and after one year. Conclusion: Our results revealed that the prevalence and incidence of HSV-2 infection are low in our HIV cases, and it is negligible in the control group. However, it seems that HIV/HSV2 co-infection has no role on HIV infection acceleration.