Iranian Biomedical Journal
Year:2004 | Volume:8 | Issue:1|Papers Count:8

The aim of this study was to isolate mouse embryonic stem cells from late blastocyst stage embryos and to use them as a model system for the study of hematopoietic induction outside the embryo by coculturing of embryonic stem cells with bone marrow stromal cells. Blastocyst stage embryos from pregnant NMRI mice were obtained and cultured for 1-2 days in DMEM medium. The inner cell masses formed colonies 4-6 days after embryo hatching they were collected and trypsinized. Several subcultures were prepared in the medium containing 0.1 mM 2 mercaptoethanol, 1000 U/ml leukemia inhibitory factor and 10% fetal bovine serum. The embryonic stem cells were recognized by alkaline phosphatase histochemistry. Embryonic stem cells were cultured on inactivated mouse bone marrow stromal cells for 3 weeks at 37 degrees C and 33 degrees C. The colony assay was performed on semisolid medium containing murine IL-3 and IL-6 to determine the differentiation of embryonic stem cells to hematopoietic progenitor cells in vitro. Our results showed that with the effect of bone marrow stromal cells, a highly pluripotent stem cell which is derived from blastocyst stage embryo was able to primarily differentiated into the hematopoietic progenitor cells. CFU-GM like colonies were recognized according to their morphology after culturing the embryonic stem cells on the condition medium supplemented with IL-3 and IL-6.

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 mM) has an apoptotic effect on MCF-7 cells and that its intracellular phosphorylation is necessary.

Brucellosis, caused by Brucella spp., is an important zoonotic disease that causes abortion and infertility in cattle and undulant fever in humans. Various studies have examined cell-free native and recombinant proteins as candidate protective antigens in animal models. Among Brucella immunogenes, antigen based on ribosomal preparation has been widely investigated. In this study, the immunogenic ribosomal protein L7/L12 gene from Brucella abortus, S19, was amplified by PCR and sub-cloned to prokaryotic expression vector pET28a. Escherichia coli BL21 (DE3) pLysS was transformed with pET28a-L7/L12 and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography with Ni-NTA resin. The concentration of purified recombinant protein calculated to 8 mg/L of initial culture. The integrity of product was confirmed by Western-blot analysis using a standard rabbit anti Brucella abortus ribosomal protein L7/L12 antibody. Sera reactivity of five infected individual were further analyzed against the recombinant ribosomal L7/L12 protein. Data indicated that recombinant ribosomal L7/L12 protein from Brucella abortus was recognized by patient sera

Herpetosiphon giganteus is a filamentous gliding bacterium. Gliding motility is the movement of the cells over surfaces without the aid of flagella. The mechanism responsible for bacterial gliding motility has not been known and there are only a few data on Herpetosiphon giganteus. The aim of this study was to observe the ultrastructure and negative staining of isolated strains of Herpetosiphon giganteus to find any organelles of locomotion. First, 35 strains of gliding bacteria were isolated from soil, freshwater, mud and activated sewage sludge. Then, 8 strains very closely related to Herpetosiphon giganteus were used for further examination. For extracellular slime and fibril observation, photoelectron micrographs were taken from different patterns on the cell surface of strains that were negatively stained. Thin sections with and without lysozyme treatment were prepared and examined by transmission electron microscopy. When the filaments were negatively stained, fibrils were detected in young cultures. There were two different kinds of fibrils in this study. The extracellular slime of these organisms was clearly visible. Examination with the electron microscopy revealed neither flagella nor an axial filament of any kind. There was no evidence for external organelles of locomotion. The results indicated that ring like structure localized at the cell surface connected with fibrils is responsible for gliding movement. The secretion of slime is necessary for adhesion of the cell to a solid surface and for ease of movement.

A Pseudomonas aeruginosa mutant derived by random mutagenesis with N-methyl-N¢-nitro-N-nitrosoguanidine, producing high levels of the rhamnolipid biosurfactants was selected on Siegmund-Wagner (SW) plates. The mutant designated P. aeruginosa PTCC1637 produces rhamnolipids at concentrations 10 time more than parent strain. NMR analysis and surface tension measurement showed that the biosurfactants produced by the mutant were identical to those produced by the wildtype strain. The biosurfactants exhibited a low surface tension of 28.0 mN m-1 and a low critical micelle concentration of 9 mg l-1. Similar to the wildtype strain, the mutant produced biosurfactants at the stationary phase

Polyethylenimine (PEI) has been proposed as a non-viral vector, and has been successfully used to transfer reporter genes into the central nervous system (CNS), kidneys, and lungs of adult mice. Neuropeptide Y (NPY) is a peptide expressed in the hypothalamus and is important in the regulation of body weight. Using PEI combined with stereotactic microinjection, we have successfully transferred cDNA-encoding NPY driven by the cytomegalovirus (CMV) promoter into the arcuate nucleus of adult male Wistar rats. Animals treated with NPY expressing plasmids (pNPY) gained more weight than the controls (p<0.05), with associated increases in food intake (p<0.05) and decreased brown adipose tissue activity, measured by Guanosine Diphosphate (GDP) binding to mitochondria, (p<0.05). In a separate study, hypothalamic slices from the rats treated with pNPY/PEI showed increased NPY release (pNPY 9.7 ± 0.3 fmol/l vs. control 8.3 ± 0.5 fmol/l, p<0.05, n = 3). These results suggest that PEI is an effective vector for gene transfer into the rodent brain and can increase the protein production sufficient to result a persistent phenotypic change. This technique offers the potential of a simple and effective method to manipulate gene expression localised to specific regions of the adult rodent brain.

A proportion of healthy neonates and adults fail to develop a protective antibody response to recombinant hepatitis B (HB) vaccine. Unresponsiveness to vaccination could be attributed to defect in a number of immunological regulatory mechanisms. In this study, IL-12 was quantitated in culture supernatant following in vitro stimulation of peripheral blood mononuclear cells isolated from a group of responder and non-responder neonates. Our results indicate significantly decreased production of HBsAg-induced IL-12 in non-responder subjects compared to responders (P<0.01). Since IL-12 is produced mainly by antigen presenting cells (APC) and is considered to be crucial for initiation and polarization of CD4+ T-cell function, therefore, our findings could be interpreted to imply APC dysfunction in non-responder vaccinees. Iran. Biomed.

In this study, urease production was investigated among thirteen strains of Aspergillus niger; seven strains isolated from soils of Semnan province in Iran and six strains obtained from Persian Type Culture Collection (PTCC). The enzyme production was screened in two submerged media quantitatively. The registered PTCC 5011 and the native S31 strains showed more urease production than the other eleven strains. The maximum enzyme productions by PTCC 5011 and S31 strains were 106 and 109 U.g-1dry mass in submerged culture, respectively. Also, we used two solid media for screening all of the strains for urease production semi quantitatively. Due to the acceptable correlations between the two methods, the latter can be used as an ancillary method to mass screening of urease production by filamentous fungi.