Iranian Journal of Basic Medical Sciences
Volume:16 Issue: 8, Aug 2013

Infection by high-risk papillomavirus is regarded as the major risk factor in the development of cervical cancer. Recombinant DNA technology allows expression of the L1 major capsid protein of HPV in different expression systems, which has intrinsic capacity to self-assemble into viral-like particles (VLP). VLPS are non-infectious, highly immunogenic and can elicit neutralizing antibodies. VLP-based HPV vaccines can prevent persistent HPV infections and cervical cancer. In this study recombinant HPV-16 L1 protein was produced in Sf9 insect cells and VLP formation was confirmed. Complete HPV-16 L1 gene was inserted into pFast HTa plasmid and transformed into DH10BAC Escherichia coli containing bacmid and helper plasmid. The recombinant Bacmid colonies turned to white and non-recombinant colonies harboring L1 gene remained blue in the presence of X-gal and IPTG in colony selection strategy. To confirm the recombinant bacmid production, PCR was applied using specific L1 primers. To produce recombinant baculovirus, the recombinant bacmid DNA was extracted and transfected into Sf9 cells using Cellfectin. The expression of L1 in Sf9 cells was identified through SDS-PAGE and western blot analysis using specific L1 monoclonal antibody. Self-assembled HPV16L-VLPs in Sf9 cells was confirmed by electron microscopy. The recombinant protein L1 was predominantly ~60 KD in SDS-PAGE with distinct immunoreactivity in western blot analysis and formed VLPS as confirmed by electron microscopy. Application of recombinant baculovirus containing HPV-16 L1 gene will certainly prove to be a constructive tool in production of VLPs for prophylactic vaccine development as well as diagnostic tests.

Methylsulfonylmethane (MSM) is a sulfur-containing compound found in a wide range of human foods including fruits, vegetables, grains and beverages. In this study the effect of MSM pretreatment on acetaminophen induced liver damage was investigated. Male Sprague Dawley rats were pretreated with 100 mg/kg MSM for one week. On day seven rats were received acetaminophen (850 mg/kg, intraperitoneal). Twenty-four hours later, blood samples were taken to determine serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Tissue samples of liver were also taken for the determination of the levels of malondialdehyde (MDA); total glutathione (GSH), superoxide dismutase (SOD), and myeloperoxidase (MPO) activity together with histopathological observations. High dose of acetaminophen administration caused a significant decrease in the GSH level of the liver tissue, which was accompanied with a decrease in SOD activity and increases in tissue MDA level and MPO activity. Serum ALT, AST levels were also found elevated in the acetaminophen-treated group. Pretreatment with MSM for one week was significantly attenuated all of these biochemical indices. Our findings suggest that MSM pretreatment could alleviate hepatic injury induced by acetaminophen intoxication, may be through its sulfur donating and antioxidant effects.

The aim of this study was to evaluate the final outcome of nerve regeneration across the eggsell membrane (ESM) tube conduit in comparison with autograft. Thirty adult male rats (250-300 g) were randomized into (1) ESM conduit, (2) autograft, and (3) sham surgery groups. The eggs submerged in 5% acetic acid. The decalcifying membranes were cut into four pieces, rotated over the teflon mandrel and dried at 37°C. The left sciatic nerve was surgically cut. A 10-mm nerve segment was cut and removed. In the ESM group, the proximal and distal cut ends of the sciatic nerve were telescoped into the nerve guides. In the autograft group, the 10 mm nerve segment was reversed and used as an autologous nerve graft. All animals were evaluated by sciatic functional index (SFI) and electrophysiology testing. The improvement in SFI from the first to the last evalution in ESM and autograft groups were evaluated. On days 49 and 60 post-operation, the mean SFI of ESM group was significantly greater than the autograft group (P< 0.05). On day 90, the mean nerve conduction velocity (NCV) of ESM group was greater than autograft group, although the difference was not statistically significant (P> 0.05). These findings demonstrate that ESM effectively enhances nerve regeneration and promotes functional recovery in injured sciatic nerve of rat.

Being secreted by the pineal gland, melatonin induces cell proliferation in normal cells and induced apoptosis in cancer cells. The purpose of this study was to investigate effects of melatonin on main components and the expression of apoptotic genes in vitrified-thawed testicular germ cells of 6- day–old mice. Testes of neonate Balb/c mice were vitrified- thawed under standard condition with or without the addition of 100 μM melatonin to both vitrification and thawing solutions. Subsequently, Vitrified-thawed wholetestes were digested under standard condition and spermatogonial stem cells type A were separate in the suspension with CD90.1 (Thy1.1+) micro beads. Extraction of RNA and synthesis of cDNA was performed. Expression levels of apoptotic genes (Fas, P53, BCL-2 and BAX) were determined using Real-time PCR. With all genes being expressed, level of expression for Fas was higher and for that of P-53 was lower than the remaining genes. Melatonin may cause apoptosis in cells being damaged under the influence of freezing thawing process. In order to examine the exact effects of melatonin on spermatogonia stem cells apoptosis, additional studies are required.

The structure- activity relationship of a series of 36 molecules, showing L-type calcium channel blocking was studied using a QSAR (quantitative structure–activity relationship) method. Structures were optimized by the semi-empirical AM1 quantum-chemical method which was also used to find structure-calcium channel blocking activity trends. Several types of descriptors, including electrotopological, structural and thermodynamics were used to derive a quantitative relationship between L-type calcium channel blocking activity and structural properties. The developed QSAR model contributed to a mechanistic understanding of the investigated biological effects. Multiple linear regressions (MLR) was employed to model the relationships between molecular descriptors and biological activities of molecules using stepwise method and genetic algorithm as variable selection tools. The accuracy of the proposed MLR model was illustrated using cross-validation, and Y-randomisation -as the evaluation techniques. The predictive ability of the model was found to be satisfactory and could be used for designing a similar group of 1,4- dihydropyridines, based on a pyridine structure core which can block calcium channels.

More than 1500 registered mutations in cystic fibrosis transmembrane regulator (CFTR) gene are responsible for dysfunction of an ion channel protein and a wide spectrum of clinical manifestations in patients with cystic fibrosis (CF). This study was performed to investigate the frequency of a number of well-known CFTR mutations in North Eastern Iranian CF patients. A total number of 56 documented CF patients participated in this study. Peripheral blood was obtained and DNA extraction was done by the use of routin methods. Three steps were taken for determining the target mutations: ARMS-PCR was performed for common CFTR mutations based on previous reports in Iran and neighboring countries. PCR-RFLP was done for detection of R344W and R347P, and PCR-Sequencing was performed for exon 11 in patients with unidentified mutation throughout previous steps. Samples which remained still unknown for a CFTR mutation were sequenced for exon 12. Among 112 alleles, 24 mutated alleles (21.42%) were detected: ΔF508 (10.71%), 1677delTA (3.57%), S466X (3.57%), N1303K (0.89%), G542X (0.89%), R344W (0.89%), L467F (0.89%). Eight out of 56 individuals analyzed, were confirmed as homozygous and eight samples showed heterozygous status. No mutations were detected in exon 12 of sequenced samples. Current findings suggest a selected package of CFTR mutations for prenatal, neonatal and carrier screening along with diagnosis and genetic counseling programs in CF patients of Khorasan.

3,4-Methylenedioxymethamphetamine (MDMA) is one of the most popular drugs of abuse in the world with hallucinogenic properties that has been shown to induce apoptosis in liver cells. The present study aimed to investigate the effects of pentoxifylline (PTX) on liver damage induced by acute administration of MDMA in Wistar rat. Animals were administered with saline or MDMA (7.5 mg/kg, IP) 3 times with 2 hr intervals. PTX (200 mg kg, IP), was administered simultaneously with last injection of MDMA in experimental group. The concomitant administration of pentoxifylline and MDMA decreased liver injury including apoptosis, fibrosis and hepatocytes damages. Our results showed for the first time that PTX treatment diminishes the extent of apoptosis and fibrosis caused by MDMA in rat liver.

The role of the Apoptosis repressor with caspase recruitment domain (ARC) in apoptosis and in certain hypertrophic responses has been previously investigated, but its regulation of Endothelin-1 induced cardiac hypertrophy remains unknown. The present study discusses the inhibitory role of ARC against endothelin–induced hypertrophy. In present study Endothelin treated cardiomyocytes were used as a hypertrophic model, that were subsequently treated with adenovirus ARC and its mutant at different multiplicity of infections. Casein-kinase-2 inhibitors were used to produce dephosphorylated ARC and to study its effect on hypertrophy. Hypertrophy was assessed by cell surface area measurement, Atrial-natriuretic-Factor mRNA analysis and total protein assay. Reactive oxygen species analysis was carried out using the dichlorofluorescin-diacetate (DCFH-DA) assay. Over expression of ARC significantly inhibits Endothelin–induced cardiomyocyte hypertrophy. The nonphosphorylated mutant ARC (T149 A) remained unable to control endothelin–induced hypertrophy, suggesting a vital role for ARC phosphorylation in regulation of its activity. Sensitization study has been carried out to check the role of endogenous ARC using casein-kinase inhibitors. Finally, the significant role of ARC in regulating reactive oxygen species -mediated control of endothelin induced hypertrophy has also been assessed. Conclusively, present study showed the vital and potential therapeutic interventional role of ARC in preventing endothelin-1–induced cardiomyocyte hypertrophy. The regulation of hypertrophic pathway by ARC relies on blunting the reactive oxygen species attack. This study further suggests a mediatory role of casein-kinase-2 in Endothelin–induced hypertrophy, mainly through its phosphorylation of ARC.

Apoptosis is a tightly regulated process and plays a crucial role in autoimmune diseases. Because abnormalities in apoptosis are considered to be involved in the pathogenesis of systemic lupus erythematosus (SLE), in present study we studied the apoptosis in T lymphocytes from Iranian SLE patientsat protein and gene expression levels for some molecules which are involved in apoptosis pathways. Thirty five SLE patients (23 female, 12 male), and 20 age matched controls (10 female, 10 male) participated in this study. T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) using MACS method. Apoptosis rate was studied at protein level by flow cytometer using Annexin V, and at gene expression level using semi-quantitative RT-PCR method for detection of Fas, FasL, Bcl-2, caspase 8, and caspase 9 genes. The percentage of apoptotic cells in SLE patients was not different in comparison with controls (20.2% ± 1.4 vs 21.1% ± 1.0), but the expression levels of FasL, caspase 8, and caspase 9 genes in all SLE patients and in female patients were significantly lower than controls; 0.45R vs 0.78R for FasL, 0.74R vs 1.0R for caspase 8, and 0.76R vs 1.26R for caspase 9 in all SLE patients and 0.37R vs 0.82R for FasL, 0.45R vs 1.6R for caspase 8, and 0.63R vs 1.56R for caspase 9 in female patients. The expression levels of FasL, caspase 8 and caspase 9 molecules involved in apoptosis decreased in female, but not in male SLE patients.

Atherosclerosis is a chronic immune-inflammatory disease that generally leads to ischemic heart disease. Ghrelin has several modulatory effects on cardiovascular system. In this study, we investigated the effect of ghrelin on aortic intima-media thickness, size and the number of adipocyte cells in obese and control mice. This study was conducted on 24 male C57BL/6 mice. The animals were divided into four groups: control, obese (received high fat diet), control+ghrelin (injected with 100 µg/Kg subcutaneously, bid) and obese+ghrelin (n=6 each). After 10 days, animals were sacrificed and epididymal adipose tissue and thoracic aortae were removed. Adipocyte cell number, size and aortic intima-media thickness were evaluated. Ghrelin did not change adipocyte cell number and size and aortic intima-media thickness in obese and control mice. In this study, high fat diet significantly decreased the number of adipocyte cells while increased their size (P<0.05). Ghrelin administration had no significant effect on adipocyte cell number and size in obese and control groups (P >0.05). In addition, it could not alter aortic intima-media thickness in both groups. Although ghrelin has several cardiovascular effects, it seems that it could not alter the size and number of adipocyte cells and aortic intima-media thickness in diet-induced obese mice

Denaturing high performance liquid chromatography (DHPLC) is a high throughput approach for screening DNA sequence variations. To assess oven calibration, cartridge performance, buffer composition and stability, the WAVE Low and High Range Mutation Standards are employed to ensure reproducibility and accuracy of the chromatographic analysis. The purpose of this study was to provide a cost-effective homemade mutation standard for DHPLC analysis. DHPLC was performed to evaluate different elution temperatures of a 374 bp DNA fragment with C>A mutation at position of 59 to achieve a peak profile similar to the Low Mutation Standard. In order to verify the reproducibility of the homemade mutation standard using DHPLC, 15 different experiments were performed to compare the homemade mutation standard, the WAVE Low Range Mutation Standard with a positive DNA control sample. We identified a comparable elution temperature and a peak profile with the WAVE Low Range Mutation Standard. This study confirmed the reproducibility of the peak profile of our homemade mutation standard compared to the Low Mutation Standard using DHPLC analysis.

Coronary artery disease (CAD) which may lead to myocardial infarction (MI) is a complex one. Great effort has been devoted to identification of genes that increase susceptibility to CAD or provide protection. A 21-bp deletion in the MEF2A gene, which encodes a member of the myocyte enhancer factor 2 family of transcription factors, has been reported in patients of a single pedigree that exhibited autosomal-dominant inheritance of CAD. Subsequent analysis of genetic variants within the gene in CAD and MI case-control settings produced inconsistent results. Here, we aimed at assessing the contribution of MEF2A to CAD in a cohort of Iranian CAD patients. Exon 11 of MEF2A wherein the above mentioned 21-bp deletion and a polyglutamine (CAG)n polymorphism are positioned was sequenced by the dideoxy-nucleotide termination protocol. In 52 CAD patients from 12 families (3-7 affected members per family) and 76 Iranian control individuals. All exons of the gene were sequenced in 10 patients and 10 controls. The 21-bp deletion was observed neither among the patients nor the control individuals. Four alleles of the polyglutamine (CAG)n polymorphism were found, but there were no significant differences in allelic frequencies between patients and controls. Sequencing of all exons of MEF2A revealed the presence of 12 novel sequence variations in introns and flanking regions of MEF2A gene, not associated with disease status. Our data do not support a role for MEF2A in coronary artery disease in the Iranian patients studied