سال:1390 | دوره: | شماره: |تعداد مقالات:6

Stress is a threatening factor that all living organisms encounter throughout life. Depending on the type of stress, there are several mechanisms for keeping body homeostasis to minimize stress effects. Brain is an organ which shows high sensitivity to stress conditions. Although many studies have shown induced-stress effects on rat embryos, little is known about the mechanisms involved in coping with stress by female rats during pregnancy. In the present study, restraint stress method was applied because this technique has been widely used in animal models to induce both psychological and physical stress.Restraint stress was applied in regular sessions (1 and 3hrs) in two groups of 6 pregnant Wistar rats and similar number of animals was used as control group receiving no stress. ACTH and corticosterone levels in plasma samples were shown to increase in response to stress treatments. On the last day of pregnancy, rat hippocampus from the brain of each animal in all three groups was removed and analyzed using 2 Dimensional Gel Electrophoresis (2DE) technique. Using Image Master Software, approximately 2000 proteins were detected in the 2D gels analyzed, among which 34 proteins exhibited differential expression. These results indicate that the proteome patterns from the hippocampus of pregnant rats subjected to 1 and 3hr of stress differs significantly from the control (unstressed) group. Future mass spectrometry identification of the 34 protein spots discovered in this study should allow a more precise understanding of molecules and cellular pathways involved in stressinduced responses during pregnancy.

Microtubules are unique cytoskeletal structures that have structural subunits of ab tubulin. Taxol is a typical microtubule stabilizing drug. The epothilones are other natural products with similar mechanism of action totaxol. Despite the highly conserved nature of b-tubulin, some organism like Saccharomyces cerevesia (S.cerevesia) is resistance to taxol, but sensitive to epothilones. In order to find differences in sensitivity of yeast tubulin to these molecules, we investigated binding mode of the taxol and epothilone A to yeast tubulin using molecular modeling. The multiple sequence alignment of b-tubulin of different species was performed using ClustalW2. Protein structure of yeast b-tubulin was constructed with Swiss Model 8.05 by using 1TVK. Modeled tubulin was superimposed with PyMol on1JFF for comparison of three-dimensional structure of two proteins. Our results showed that one of the most interesting differences in binding mode of these molecules is residue 227. The His 227 in bovine makes a hydrogen bond by means of its d-nitrogen with epothilone A and by means of its e-nitrogen with taxol. The Asn227 of yeast can play role of the d-nitrogen of imidazole ring of H227, but not of e-nitrogen of it. So yeast tubulin in contrast to taxol can interact with epothilone A. Due to conservation of essential residues for binding (T274, R282 and Q292), epothilone A in comparison with taxol can tolerate the interchange in the binding pocket (R276I). Our findings may be of a great aid in the rational design of antitumor agents that bind to the taxol binding region of tubulin.

In many acute leukemias, normal differentiation does not occur. However, in many cell lines derived from hematologic malignancies, differentiation or apoptosis can be induced by variety of agents. Despite advances in the treatment of Acute Lymphoblastic Leukemia (ALL), in most patients long-term survival rates remain unsatisfactory, especially in T-cell derived ALL. Thus we studied the anticancer effects of fenretinide, 1a, 25 (OH)2D3, and bryostatin-1 in CCRF-CEM (T-cell derived) and Nalm-6 (B-cell derived) ALL cell lines. Using MTT assays, both cell lines were shown to exhibit increased inhibition of proliferation at micro (fenretinide) and nanomolar (1a, 25 (OH)2D3, bryostatin-1) concentrations. These anti-cancer agents were shown to induce apoptosis and activate caspase-3 pathway in both ALL cell lines. Furthermore, for the first time we are reporting consistent anti-proliferative and apoptotic effects of Bryostatin-1 in ALL T-cell derived cell line with the lowest ED50 (ranging 4.6-7.4 nM). To evaluate the differentiation induction by fenretinide, 1a, 25 (OH)2D3, and bryostatin-1 in ALL cell lines, we assayed for the expressions of CD19, CD38 markers on Nalm-6 and CD7 marker on CCRF-CEM cell line. The flow cytometric analysis showed a significant increase in expression of CD markers in response to anticancer drug treatments. To assay the effects of anti-cancer drugs on cell cycle distribution, cell cycle analysis using flow cytometry was employed. These anti-cancer drugs appear to affect the CCRFCEM and Nalm-6 cell cycles differently (Go/G1 and G2/M arrest, respectively).Overall results demonstrate that the anticancer agents used in this study are strong inhibitors of ALL cell proliferation and inducers of apoptosis and differentiationin vitro. These findings may be quite helpful if these drugs are to be used for dif-ferentiation therapy of ALL patients in clinics in the future. Further studies are warranted to establish thein vivo effect of these drugs particularly in patients with T-cell derived ALL.

Today, there is an increasing interest in the use of metal nanoparticles in health sciences. Amongst all nanoparticles, the gold nanoparticles have been known to kill the cancer cells under hyperthermic condition by near-infrared frequency electromagnetic waves. On the other hand, although there are different physiochemical methods for disinfection of microbial pollution, however applications of irradiated gold nanoparticles against microorganisms have not yet been investigated. In this study, gold nanoparticles were prepared using D-glucose and characterized (particle size<26 nm). In the next step, the enhancing effect of the non toxic level of gold nanoparticles (50 mg/mL) on the antimicrobial activity of 2450 MHz electromagnetic radiation generated at a microwave oven operated at low power (100 W), was investigated by time kill course assay against Staphylococcus aureus (S.aureus) ATCC 29737. The results showed that application of gold nanoparticles can enhance the lethal effect of low power microwave in a very short exposure time (5 s).

In previous years, identification of fetal cells in maternal blood circulation has caused a new revolution in non-invasive method of prenatal diagnosis. Low number of fetal cells in maternal blood and long-term survival after pregnancy limited the use of fetal cells in diagnostic and clinical applications. With the discovery of cell-free fetal DNA (cffDNA) in plasma of pregnant women, access to genetic material of the fetus had become possible to determine early gender of a fetus in pregnancies at the risk of X-linked genetic conditions instead of applying invasive methods. Therefore in this study, the probability of detecting sequences on the Y chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women at 6th to 10th weeks of gestation and then the fetal DNA was extracted from the plasma. Nested PCR was applied to detect the sequences of single copy SRY gene and multi copy DYS14 & DAZ genes on the Y chromosome of the male fetuses. At the end, all the obtained results were compared with the actual gender of the newborns. In 40 out of 42 born baby boys, the relevant gene sequences were identified and 95.2% sensitivity was obtained. Non-invasive early determination of fetal gender using cffDNA could be employed as a pre-test in the shortest possible time and with a high reliability to avoid applying invasive methods in cases where a fetus is at the risk of genetic diseases.

Expressions of recombinant proteins for different applications are important objectives in molecular biotechnology, however, expression of some recombinant proteins is difficult. Several methods have been designed for expression of these proteins. The aim of this study was to construct a vector containing Mtb32C fragment of Mycobacterium tuberculosis (M.tuberculosis) as a fusion partner in order to improve the expression of fused recombinant proteins. Mtb32C was amplified by polymerase chain reaction (PCR). The amplified fragment was ligated into pET21b+vector. Colony-PCR, enzyme digestion and DNA sequencing methods were used to confirm the recombinant vector. Colony-PCR showed a 420bp fragment in size corresponding to the correct size of our fragment. In addition the recombinant plasmids sequencing showed the accuracy of the cloned fragment. For confirming the expression, reverse transcriptase (RT)-PCR analysis was performed showing a 420 bp fragment in agarose gel electrophoresis using specific primers. The construction of a vector containing Mtb32C fragment is promising as a fusion partner for future studies as it affected the expression of the fused proteins and increased immune responses against the partner.