Journal of Pathobiology Reaearch
Volume:15 Issue: 1, 2012

The aim of the present study was the production of recombinant lentviruses that express miR-16. After transduction, altered expression levels of miRNA and its target protein were analyzed. A DNA fragment that contained the miR-16 precursor was cloned in a lentiviral plasmid. Lentiviral vector particles were produced by transient calcium phosphate co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids. Viral supernatants were harvested and concentrated by ultracentrifuge. Virus titration was determined by fluorescent microscopy and flow cytometry. Altered expression levels of miR-16 were evaluated by real-time PCR; its protein target was evaluated by Western blot. The identity of DNA was established by colony-PCR, enzymatic digestion of positive clones, and DNA sequencing. After co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids, viral particles were concentrated and the virus titer determined. Maximum expression of the GFP reporter gene was obtained in more than 80% of the cells transduced with lentivirus at MOI=1. Real-time PCR assay showed that miR-16 expression levels significantly increased in transduced cells compared with the control group. As shown by Western blot analysis, miR-16 overexpression downregulated Bcl-2 expression at the protein level. This lentivirus expression system could be considered as a tool for efficient delivery of produced miRNAs to cells.

Human cytomegalovirus (CMV) is a major life-threatening pathogen for hematopoietic stem cell transplant recipients. Specific tests are used for the diagnosis and monitoring of CMV infection in transplant patients. This study evaluates the performance of pp65 antigenemia and qualitative PCR assays for monitoring CMV in such patients. We analyzed 179 clinical samples from 41 patients by using a validated home-brewed qualitative PCR and a commercial antigenemia assay. The obtained results were evaluated using quantitative real-time PCR as the gold standard. CMV was observed in 26.8% of samples analyzed by the antigenemia assay and in 42.6% of the samples by qualitative PCR. Among 179 clinical samples, 50.8% were negative and 21.2% were positive by both assays. On the other hand, 26.3% were only positive by qualitative PCR whereas 1.7% were positive by the antigenemia assay. A comparison of the results with real-time PCR showed that qualitative PCR has a higher sensitivity than the antigenemia assay (98.7% vs. 45.7%). The specificity of both assays was equal (96.8%). Quantitative results of the antigenemia assay showed good correlation with real-time PCR (r=0.715; p<0.001). Both the qualitative PCR and antigenemia assays have special deficiencies for efficient diagnosis of CMV infection. Therefore, effective management of CMV infection in transplant patients requires the use of other sensitive quantitative methods such as qPCR.

This study was designed to investigate the antinociceptive effect of curcumin in diabetic rats by using the formalin and hot tail immersion tests. Wistar rats were divided into the following six groups: control; curcumin-treated control (50 mg/kg); diabetic; sodium salicylate (SS)-treated diabetic; and two curcumin-treated diabetic groups (10 and 50 mg/kg). Curcumin was administered seven days after streptozotocin injection for a total of five weeks. High-dose curcumin treatment of diabetic rats reduced the pain score in both acute and chronic phases of the formalin test (p<0.05). SS-treated diabetic rats had a reduction in pain score only in the chronic phase of the formalin test (p<0.05). In the hot tail immersion test, diabetic rats showed a significant reduction in tail flick latency compared to the control group (p<0.01). High-dose curcumin treated diabetic rats showed significantly increased latency relative to untreated diabetic rats (p<0.05). Diabetic rats also showed a significant increase in the tissue level of malondialdehyde (MDA; p<0.01). High-dose curcumin treated diabetic rats had a significantly reduced level of MDA (p<0.05). Chronic administration of curcumin could attenuate the nociceptive score in both the acute and chronic phases of the formalin test in a streptozotocin-induced experimental model of diabetes mellitus and increase thermal pain threshold. The beneficial effect of curcumin is partly attributed to attenuation of lipid peroxidation in the periphery.

Cutaneous leishmaniasis is an endemic infectious disease considered to be a crucial health problem in many countries, including Iran. As such, there is a need for new medications with few side effects. In the present research we have studied the effect of artimisinin on Leishmania major (L. major) and cell death in vitro. A specific number of promastigotes of L. major were grown in the presence of different concentration of artimisinin to achieve IC50 of the drug. The MTT method was applied to evaluate the cytotoxic effect of the artiminisinin on L. major. Various densities of this drug were applied to study the induction of apoptosis by flow cytometry on L. major promastigotes. We calculated the IC50 of artimisinin to be 25 μg/mlby promastigote assay. Promastigotes were incubated at 72 hours incubation with various doses of artimisinin (10, 25, 50 and 100 μg/ml). The dose 100 μg/ml showed the most apoptosis (68.16%) by Annexin-V FITC. Whereas the 10 μg/ml dose had the least apoptosis (12.78%). There was no change in the control group. According to MTT, the toxic effect of artiminisinin on L. major promastigotes increased with increasing drug concentration. This study revealed that artimisinin has a little toxic effect on macrophages. According to the flow cytometry and MTT results, artimisinin can be suggested as an appropriate drug for in vivo antileishmanial study.

Alpha-synuclein is a major component of protein plaques in synucleinopathies, particularly Parkinson’s disease. The purpose of this study is to assess the inhibitory effects of cuminaldehyde on the fibrillation of alpha-synuclein. Alpha-synuclein was expressed in Escherichia coli and subsequently purified. For the process of fibrillation, purified protein was incubated at 37◦C and pH 7.2. Fibrillation was analyzed by the standard fibril methods. The effects of different concentrations of cuminaldehyde (20-500 μM) on alpha-synuclein fibrillation were studied by assessment of the cytotoxic effects of samples on the neuroblastoma cell line, SK-N-MC. To study the protein aggregation forms that were generated in the presence of cuminaldehyde, SDS resistance and induced fibrillation (seeding) methods were employed. For studying its specificity on alpha-synuclein, the effect of cuminaldehyde on lysozyme fibrillation was also examined. We showed, for the first time, that cuminaldehyde inhibited fibrillation by more than 80%. The highest inhibition was observed at the ratio of 5-15 moles of drug to protein. The viability of the treated cells with inhibited proteins was more than 90%, whereas non-inhibited samples caused a decrease in viability by 50%. Inhibited samples were not resistant to SDS and they were unable to induce fibrillation. Cuminaldehyde did not inhibit lysozyme fibrillation. Cuminaldehyde inhibited fibrillation of alpha-synuclein which was accompanied by small amorphous aggregated particles of alpha synuclein. The inhibited protein samples did not induce aggregation. Thus, cuminaldehyde can be considered as a candidate to inhibit the formation of alpha-synuclein plaques.

Organophosphorus hydrolase (OPH) is a homodimeric enzyme that can hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. This makes OPH a suitable element for the biodegradation of these compounds. We successfully cloned the OPH gene from Pseudomonas diminuta, after optimization for Pichia pastoris, into a yeast expression vector (pPICZαB). After transformation and induction of recombinant yeasts, the expressed enzyme was investigated for its biochemical and kinetical parameters. The enzyme was purified 7.49-fold to a specific activity of 0.421×103 U/mg protein from the supernatant with a yield of 33%. The purified enzyme was able to degrade organophosphates. It had an optimal activity and stability up to 50°C, and a pH range of 7.0-10.0. The enzyme had a Km of 45.96 µM and a Vmax of 11.23 µM/min (421 µM/min/mg) for paraoxon as a substrate. This enzyme was sensitive to divalent cations and inactivated by denaturing compounds such as SDS. The molecular mass of the purified enzyme as estimated by SDS–PAGE analysis was approximately 40 kDa. In this study, the purified enzyme effectively hydrolyzed paraoxon, an organophosphorus compound. The activity and stability of this enzyme at high temperatures and pH, and low Km in comparision with bacterial isolates could make it an attractive biocatalyst for applied bioremediation and biosensing.

Toxoplasmosis is a worldwide disease, for which different detection methods have been used. The nucleic acid sequence-based amplification (NASBA) method is shown to be highly efficient for diagnosis of live microorganisms. The present research evaluates the molecular isothermal method of NASBA to identify live Toxoplasma gondii (T. gondii) in rat. Tachyzoites of T. gondii were inoculated in the peritoneal cavities of mice (Mus musculus) and their RNA was extracted. The NASBA method was then used to amplify the tachyzoite B1 rRNA gene. Next, we examined blood samples from 30 experimentally infected case and control rats (Rattus norvegicus) by NASBA. Finally, the resultant band was investigated on an agarose gel. The B1 genes extracted from both the tachyzoites and blood samples were successfully amplified by the NASBA method. This amplified gene yielded an amplicon of approximately 116 bp on gel agarose. NASBA is highly efficient for the identification of live T. gondii. This method can be applied for early diagnosis of active toxoplasmosis in both newborns and immunocompromised individuals.

GBV-C is a virus transmissible via blood transfusion and sexual routes. Because of the shared transmission routes in GBV-C and hepatitis B virus (HBV) we have attempted to detect GBV-C in HBsAg-positive patients using the more sensitive semi-nested PCR. We used restriction fragment length polymorphism (RFLP) to determine genotype. A total of 100 serum samples were collected from HBsAg-positive patients from 1389-1390. After designing specific primers and optimizing the semi-nested PCR, sequences of PCR products were analyzed by Neb Cutter software. Restriction enzyme sites were determined and suitable enzymes selected for RFLP. Semi-nested PCR shows acceptable sensitivity for the detection of GBV-C and its genotype determination. This technique is more affordable than other techniques. There appears to be a high rate of GBV-C infection among Iranian HBV patients. In this study, the majority of patients co-infected with GBV-C were of HBV genotype 2, which is similar to the pattern seen in the US and Europe.