Iranian Biomedical Journal
Volume:10 Issue: 3, Jul 2006

Human embryonic stem cells (hESC), which are derived from the inner cell mass of the blastocysts, have been considered to be pluripotent cells. In this study we examine the differentiating potential of hESC into hepatocytes by characterization of the expression of endoderm and liver-specific genes. hESC were cultivated in suspension to form aggregates, the embryoid bodies. They were allowed to outgrowth on the plated culture with the stepwise addition of growth factors such as acidic fibroblast growth factor (aFGF), hepatocyte growth factor and oncostatin M into the culture medium. The expressions of endodermal and liver specific genes such as hepatocyte nuclear factor 3β, alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK-8), CK-18, transthyretin, glucose 6-phosphatase and tyrosine aminotransferase were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The expressions of ALB and CK-18 in the cytoplasm were analyzed by Immunohistochemistry. The immunoblotting and chemiluminescence of the conditioned media indicated the secretion of ALB and AFP. RT-PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of hESC. Our results showed the expression of endoderm and hepatic specific genes after in vitro differentiation of hESC into hepatocyte-like cells through addition of various growth factors in three dimensional culture systems (collagen type I). hESC could be a new potential source of hepatocyte for transplantation in patients with liver failure. Iran. Biomed.

Norepinephrine plays a trophic role in the control of cell replication and differentiation in target cells that express adrenergic receptors. In this study, we have tested the influence of infraphysiological, physiological and supraphysiological concentrations (0.0001 nM, 1 nM, 10000 nM) of human norepinephrine on the proliferation of breast cancer cells (human breast adenocarcinoma [MCF-7]) in co-culture with human adipocytes in three-dimensional collagen gel matrix culture. Cell proliferation and lipolysis rate were measured by 3-(4, 5-dimethylthiazolyl)-2, 5-diphenyl-tetrazolium bromide (MTT) and Oil red O colorimetric assay in second, 7th and 14th days of culture experiments. Our results showed a direct correlation between lipolysis rate of adipocytes and proliferation rate of MCF-7 cells. Both physiological and supraphysiological concentrations of human norepinephrine significantly (P<0.05) increased the proliferation of MCF-7 cells synchronously with progress of adipocyte lipolysis. The proliferations of MCF-7 cells were significantly decreased after conversion of adipocytes to fibroblast-like cells by supraphysiological concentration of norepinephrine. There was no statistical difference in lipolysis of adipocytes and proliferation of MCF-7 cells in response to infraphysiological concentration of norepinephrine. These findings indicated that norepinephrine stimulated the proliferation of MCF-7 cells in co-culture with human adipocytes as a lipolytic factor and that norepinephrine effect was suppressed by conversion of adipocytes to fibroblast-like cells, suggesting adipocytes as another target for prevention and therapy of breast cancer. Iran. Biomed.

Silk fibroin is a suitable protein for osteogenesis by inducing markers of bone formation in the cultures of osteoblasts, so we examined the ability of this protein to induce mineralized nodules in the rat bone marrow stromal cell cultures. Bone marrow stromal cells obtained from 4 to 6 weeks old Spruge-Dawely male rats were grown in primary culture for seven days and then subcultured for 21 days. The secondary cultures were done on either silk fibroin-coated polystyrene plates or free-silk fibroin ones. After 21 days of grow up, the cultures were examined for nodule formation by scanning electron microscopy, for mineralization by alizarin red S staining and for expression of gene markers of osteoblast maturation by reverse transcription PCR (RT-PCR). The stromal cells were observed to form three-dimensional nodules when cultured on the silk fibroin and compared to the stromal cells cultured on the free-silk fibroin polystyrene plates, where no nodules were observed in the time-frame studied. These nodules were also found to be mineralized and expressed the gene markers of osteoblast. Silk fibroin could serve as suitable inducing factor by stimulating stromal cell differentiation to form mineralized nodules. Iran. Biomed.

Combination of glycation and oxidation is associated with diabetes mellitus. The aim of this study was to clarify the effect of glycated proteins in presence of transition metal ions on production of reactive oxygen species (ROS) in rat hepatocyte suspension. Glycated albumin was prepared by incubation of bovine serum albumin with 100 mM glucose in 0.3 M phosphate buffer at 37°C for 2, 4, and 6 weeks. The prepared rat liver cell suspension was treated with glycated albumin in presence of either Fe+++ or Cu++ ions. Produced malone-dialdehyde was measured as an indicator of ROS and of cell injury. The results showed Fe+++ and Cu++ ions increase the ROS production in presence of glycated albumin (p<0.01). All prepared glycated albumin showed cytotoxicity in rat hepatocytes suspension in the presence of cupric and ferric ions, and this injury was dependent to metal ion concentration. Higher degree of glycation showed higher effect on ROS production (p<0.01). Comparing the effect of Fe+++ and Cu++, cupric ion had higher cytotoxic effect (p<0.01). These results indicated that hepatocytes may be damaged by ROS which are produced by the interaction of the glycated albumin and transition metal ion. Iran. Biomed.

Teucrium polium has been known as an important traditional medicinal plant and is used for different therapeutic purposes such as gastrointestinal disorders. Therefore, the anti-spasmodic and anti-nociceptive activities of aqueous extract of Teucrium polium w as examined. Anti-spasmodic effect of different concentrations (47-470 mg/l) of Teucrium polium extract was assessed on acetylcholine (220 nM) precontracted guinea pig isolated ileum. The anti-cholinergic effect of the plant was also examined by obtaining concentration-response curves in the absence and presence of Teucrium polium extract (470 mg/l) and atropine (10 nM). Anti-nociceptive effect of different doses (30-240 mg/kg) of Teucrium polium aqueous extract was determined by hot-plate test on mice and compared with the effect of morphine (10 mg/kg) as positive control. Maximum inhibition response induced by Teucrium polium extract on contraction induced by acetylcholine (220 nM) was 93.5%. In the absence and presence of Teucrium polium extract (470 mg/l) and atropine (10 nM) the EC50 (the effective concentration causing 50% of maximum response) of Ach were 28.3 ± 2.1, 55.4 ± 3.7 and 208.1 ± 9.2 nM respectively. There was also a parallel right-ward shift in the log concentration-response curve of acetylcholine in the presence of atropine, but a non-parallel shift in the presence of Teucrium polium extract. The Teucrium polium extract increased reaction time dose-dependently (P<0.01 for all doses). However the anti-nociceptive effect of extract was significantly less than that of morphine (P<0.001). These results show that Teucrium polium aqueous extract have anti-nociceptive and anti-spasmodic effects and may have some clinical benefits for gastrointestinal disorders. Iran. Biomed.

Non-steroidal anti-inflammatory drugs (NSAID) have been associated with antioxidant property and have been shown to improve the circulating antioxidant status on daily dosing in different inflammatory conditions. The present study was conducted to investigate the antioxidant role of meloxicam in aluminum induced oxidative stress in rat brain. In the in vivo experiments, Sprague-Dawley rats where randomized into 4 groups receiving daily treatment for 4 weeks: 1) double distilled water i.p., 2) 4.2 mg/kg aluminum i.p. 3) meloxicam (5.0 mg/kg, i.m.) 4) 5.0 mg/kg, meloxicam i.m. + 4.2 mg/kg aluminum i.p. brain homogenates from the above animals were assayed for lipid peroxidation levels as well as superoxide dismutase activity. In the in vitro experiments, brain homogenates from Sprague-Dawley rats were treated with either, aluminum, meloxicam or their combinations and were then assayed as per the in vivo samples. In vivo data showed elevated lipid peroxidation levels in brain homogenate in aluminium-treated group as compared to aluminium + meloxicam treatment which showed a significant decrease in the malonaldehyde levels. Similar results were observed in the in vitro experiments when brain homogenates were treated with either, aluminum, meloxicam or their combinations. Furthermore, no change was observed in superoxide dismutase activity in any treatment group as compared to the control in either experiment. These results indicate that NSAID can be used in Alzheimer management. Iran. Biomed.

Recent research on several DNA fragments covering open reading frames (ORF) 1-37 shows a new genetic marker in ORF 6 which is specific for differentiating wild-type varicella-zoster virus (VZV) strains from Oka varicella vaccine strain. On the other hand, herpes simplex virus (HSV) genome analysis by restriction enzymes is used to differentiate types one and two of the virus and even strains of each type. Previous studies using PCR-sequencing technique have shown that the thymidine kinase (TK) gene of HSV-1 is polymorphic. In this study, TK gene and DNA binding protein (UL29) gene of HSV-1 were selected. Both genes were analyzed with restriction endonucleases in order to identify a genetic marker for differentiating native strains of HSV-1 from the foreign strain. Three isolates of HSV-1 as well as standard strain of KOS were propagated in Vero cells. Initially, a pair of specific primers for each gene was designed to amplify UL29 and TK genes of these isolates. Subsequently, PCR products of these genes were digested separately with five restriction enzymes and subjected to polyacrylamide gel electrophoresis. Using PCR-RFLP (Restriction fragment length polymorphisms) technique, results indicate that the patterns of restriction endonuclease digestion of UL29 and TK genes of the three isolates show no differences when compared to KOS strain. The genotypes of Iranian isolates are the same as KOS genotype and both genotypes are derived from a common ancestor. Hence, it can be postulated that in the process of random population flow among Iran, Europe and USA, the original KOS strain infected the Iranian population at some point in time. Iran. Biomed.

The development of resistance to antimicrobial agents is a major problem in chemotherapy. Finding agents which potentiate antimicrobial activity could be favorable. There are some reports that methylxanthines changed the inhibitory effect of antibacterial agents. Thus, possible synergistic effect of methylxanthines, aminophylline and caffeine on some antibiotics, carbenicillin, ceftizoxime and gentamicin, which are effective on P. aeruginosa and Staphylococcus aureus, were studied. The interaction of methylxanthines and antibiotics were studied in vitro using a checkerboard method. At concentrations of 0.25-4 mg/ml, aminophylline and caffeine decreased the MIC of the antibiotics 2-4 times against P. aeruginosa and Staph. aureus. Both methylxanthines also reduced the minimum bactericidal concentration of the antibiotics by up to 2 times. Caffeine and aminophylline had no antimicrobial effect themselves. The results of the present study reveal that aminophylline and caffeine potentiated the antimicrobial action of carbenicillin, ceftizoxime and gentamicin against Staph. aureus and P. aeruginosa. Iran. Biomed.