Iranian Biomedical Journal
Volume:9 Issue: 4, Oct 2005

Fusion of two genes at DNA level produces a single protein, known as a chimeric protein. Immunotoxins are chimeric proteins composed of specific cell targeting and cell killing moieties. Bacterial or plant toxins are commonly used as the killing moieties of the chimeric immunotoxins. In this investigation, the catalytic domain of Shiga-like toxin (A1) was fused to human granulocyte macrophage colony stimulating factor (hGM-CSF) gene and the fused gene was then expressed using an expression vector containing arabinose promoter. The protein thus obtained could be recognized by two different ELISA system designed for detection of hGM-CSF and Shiga-toxin and reconfirmed by Western-blot. The recognition of the chimeric protein by specific antibodies could be indicative of the proper form of the protein, which justifies further steps to be taken to evaluate the potential effects of the chimeric protein..

Protease 2A (2Apro) of coxsackievirus B3 (CVB3) plays a major role in viral replication. In case of infection, viral proteins are being synthesized from viral mRNA using host biosynthesis machinery. 2Apro of virus, after being synthesized, exhibits two critical functions, cleavage of viral proteins and breaking eukaryotic initiation factor 4G. The enzyme plays an essential role in viral replication and cellular damage. To understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure 2Apro enzyme. cDNA of 2Apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pET22b(+). Since 2Apro is a toxic product, its expression will act on host before induction and damages the cells. For this reason, different hosts were checked and finally BLR(DE3)pLysS, which carries an extra-plasmid for lysozyme expression, that minimizes unwanted target protein products (leakage) was selected. By employing such expression system we could minimize the unwanted expression of 2Apro. Though it is not possible to avoide it, but seems negligible. Hence, this system is useful for expression of toxic proteins in sensitive hosts in order to prevent bacterial damage. The product was confirmed by SDS polyacrylamide gel electrophoresis and immunoblot analysis.

Recombinant human interferon alpha 2a (rhIFN a-2a) production and cell growth were monitored in a set of genetically modified E. coli strains (MSD1519, MSD1520, MSD 1521, MSD 1522, MSD 1523) producing rhIFN a-2a. The growth was followed at OD 600 nm, changes in cell physiology were detected by pyrolysis mass spectrometry (PyMS) of cell biomass and recombinant protein production was determined by SDS-gel electrophoresis. The heat stress applied was minimal (50°C for 5 minutes) but the effects were detected in most of the strains. All the strains except MSD 1520 showed a significant increase in the quantity of the rhIFN a-2a secretion at 25 hour growth under the heat shock condition, quantitated by the Bio-Rad Molecular analyst software, MultiAnalyst from the digital image of gels captured using a Fluor S image analysis system. In the main fermentation system at T7 hour, only MSD 1523 showed an increase in the rhIFN a-2a secretion under the heat shock condition, at T8 hour MSD 1520 and MSD 1523 had an increased rhIFN a-2a secretion, and at T10 MSD 1521 and MSD 1522 had an increased rhIFN a-2a secretion under the heat shock condition. The PCCV ordination diagrams obtained from the PyMS result showed, a considerable effect of heat shock on the MSD 1519 strain at T5 hour. For the other strains, the result largely agreed with both the growth curves and the rhIFN a-2a production that had a limited effect on E. coli cultures. The increase of temperature in the main fermentation during the log phase of the bacterial culture during rhIFN a-2a expression depends on the strain specificity. This situation could definitely lead to over expression of the gene and higher intracellular accumulation of rhIFN a-2a molecule.

The main role of cumulus cells (CC) is to provide nutritious materials for developing oocytes. Follicular fluid (FF) contains the enzyme acid phosphatase, which plays a role in ovulation and fertilization of oocyte. The objective of this prospective study was to evaluate the levels of acid phosphatase in FF and the rate of apoptosis in CC of fertilized and unfertilized oocytes in intracytoplasmic sperm injection (ICSI) program with male factor infertility. Following ovarian hyperstimulation, 101 mature oocytes were prepared for ICSI. Fertilized (n = 51) and unfertilized (n = 50) oocytes were evaluated for apoptosis in CC. CC of each oocyte were stained with Hoechst 33258 for immunofluorescent microscopy. For evaluation of acid phosphatase, FF was centrifuged and the enzymes levels were measured with spectrophotometer. Nuclei of apoptotic cells were fragmented, the chromatins condensed, and the apoptotic bodies were observed in some cells. Rates of apoptotic CC in fertilized and unfertilized oocytes were 15.83% and 13.34%, respectively (P>0.05), r = 0.520). The acid phosphatase level was reduced as the rate of apoptosis increased P<0.05, r = -0.520). Also, the concentration of the enzyme increased when the percentage of normal CC increased P<0.05. Our results confirm that neither the evaluation of CC apoptosis, nor the level of acid phosphatase have a prognostic value in the outcome of ICSI. However, the FF level of acid phosphatase is directly related to the quality of retrieved MII oocyte..

Zn (II) is an important regulator of caspase-3, as well as an antioxidant, microtubule stabilizer, growth cofactor, and anti-inflammatory agent. Over the past 30 years, many researchers have demonstrated the important role of Zn (II) in a variety of physiological processes, including growth and development, maintenance and priming of the immune system, and in tissue repair and regeneration. In this study, we present evidence that chelation of extracellular zinc by diethylenetriaminepentacetic acid (DTPA) in different concentrations causes cell death in carcinoma cell lines, HT29/219 and SW742. Hoechst 33258 staining revealed that cell death was mainly by apoptosis. Additionally, significant increases in the activity of caspase-3 and -9 were observed in both cell lines. Caspase-8 activation was negligible in both cell lines. The cytotoxicity/apoptotic effect of DTPA was inhibited significantly by Zn (II), Cu (II) and N-Acetyl-L-Cysteine (NAC) (P<0.05). Therefore, DTPA, the membrane-impermeable metal ion chelator, induces apoptosis through the depletion of extracellular zinc ion.

It is a well-established fact that adenosine and its receptor subtypes (A1 and A2) are involved in changes of contractility, heart rate and coronary blood flow (CBF) under different circumstances. This study was conducted to evaluate the role of nitric oxide and prostaglandins in development of these changes. For this purpose, Nitro-L-Arginine methyl ester (L-NAME), and indomethacin as inhibitors of nitric oxide and prostaglandins synthesis were used respectively. In this respect, guinea pig isolated hearts were randomly divided into control (receiving adenosine) and groups II and III which received L-NAME (100 µM) and indomethacin (50 nM) before adenosine application, respectively, using isolated heart setup. The results showed that adenosine increased CBF and decreased heart rate and contractility in control group. In the presence of L-NAME, adenosine was less effective in enhancing the CBF and decreasing cardiac contractility. Furthermore, no significant change was observed in the presence of indomethacin (regarding all of parameters). It can be concluded that nitric oxide (and not prostaglandins) is essential for the effect of adenosine on CBF and cardiac contractility..

Oxygen free radicals may be implicated in the pathogenesis of ischemia reperfusion damage. As the antioxidant effects of some species of Pistacia have been reported, the protective effects of Pistacia vera L. gum extract (0.1-0.5 g/kg) on oxidative damage following cerebral ischemia were studied in rats. Ischemia was induced using four-vessel occlusion model and evaluated using measurement of malondialdehyde (MDA) and antioxidant power in hippocampus. MDA and antioxidant power were assayed with the thiobarbituric acid (TBA) and ferric reducing-antioxidant power (FRAP) tests, respectively. The extract and saline were administered intraperitoneally 10 min subsequent to ischemia. Results have shown that the MDA level increased by 47% and antioxidant power decreased by 117% in control group in comparison with sham-operated animals (P< 0.001). Treatment with P. vera L. gum extract significantly and in a non-dose-dependent manner reduced brain MDA level by 63% (P<0.001) and increased antioxidant power of brain by 235% (P<0.001) in comparison to the controls. P. vera L. gum comprised of saponins, tannins, and flavonoids. According to these results, it is suggested that P. vera gum may exhibit neuroprotective effects against ischemia..

Accurate and rapid diagnosis of human cytomegalovirus (HCMV) disease in immunocompromised patients has remained as a challenge. Quantitative competitive PCR (QC-PCR) methods for detection of HCMV in these individuals have improved the positive and negative predictive values of PCR for diagnosis of HCMV disease. In this study we used QC-PCR assay, using a co-amplified DNA standard, to quantitate the HCMV glycoprotein B (gB) gene in different samples. A DNA internal standard (IS) was designed by replacing HCMV primer binding site at 5'' ends of primers that amplifies a 156-bp fragment of lambda genome, and a 200 bp amplicon was produced. Two DNA fragments of 257 bp wild type and 200-bp (IS) were co-amplified with the same oligonucleotide primer sets, analyzed by gel electrophoresis and used for construction of a standard curve. From this, the copy number of the gB gene present in different samples could be determined. Co-amplification with 1,000 copies of IS, allowed quantitation of 10-100,000 of HCMV DNA in a single PCR. This rapid assay avoids using radioactive components and other less efficient quantitative systems. It has the potential for early identification of patients at high risk of development of HCMV disease, and is useful for therapeutic monitoring.