Iranian Journal of Basic Medical Sciences
Volume:11 Issue: 3, Autumn 2007

Apoptosis or programmed cell death is a gene regulated phenomenon which is important in both physiological and pathological conditions. It is characterized by distinct morphological features including chromatin condensation, cell and nuclear shrinkage, membrane blebbing and oligonucleosomal DNA fragmentation. Although, two major apoptotic pathways including 1) the death receptor (extrinsic) and 2) mitochondrial (intrinsic) pathway have been identified, recently endoplasmic reticulum and lysosomal pathways have been also recognized. Depending on both the cell type and the initiating factor, distinct pathways are activated. The pathways share a common final phase of apoptosis, consisting of activation of the executioner caspases and dismantling of substrates critical for cell survival. The important regulatory mechanisms include death receptors, caspases, mitochondria and Bcl-2 family proteins. Modulating of apoptosis is a novel therapeutic strategy in treatment of different diseases. These include situations with unwanted cell accumulation (cancer) and failure to diminish aberrant cells (autoimmune diseases) or diseases with an inappropriate cell loss (heart failure, stroke, AIDS and neurodegenerative diseases). Modulation of apoptosis is a novel therapeutic strategy in treatment of different diseases. Many approaches including gene therapy, antisense strategies and numerous apoptotic drugs to target specific apoptotic regulators, are currently being developed. The goal of this review is to provide a general overview of current knowledge on the process of apoptosis including morphology, biochemistry, signaling as well as a discussion of apoptosis in diseases and effective therapy.

It is believed that the mesenchymal stem cell (MSC) differentiation and proliferation are the results of activation of wnt signaling pathway. On the other hand, lithium chloride is reported to be able to activate this pathway. The objective of this study was to investigate the effect of lithium on in vitro proliferation and bone differentiation of marrow-derived MSC.In this experimental study, rat marrow cells were plated in a medium supplemented either with or without 2-10 mM lithium and expanded through three successive subcultures. To explore the impact of lithium on cell growth, doubling time (DT) of marrow cell population was determined for all the cultures. To determine the lithium effects on osteogenesis, the proliferation medium of passged-3 cells from all cultures were replaced by osteogenic media, with or without 2-12 mM lithium. Osteogenesis was then quantified by measurement of the amount of matrix mineralization and the expression of bone-specific genes.DT results indicated that the marrow cells in 4 mM lithium concentration were grown faster than the others (P<0.05). Intensive matrix mineralization and abundance of bone specific gene expression were observed in the cultures with 10-12 mM lithium concentration. All these differences were statistically significant. According to the results, all lithium -treated cultures possessed more differentiation than the control. Moreover, lithium low concentration was associated with more proliferation and its high concentration with more differentiating effects.Lithium chloride at 4 mM concentration promotes MSC proliferation and at 10-12 mM enhances MSC osteogenic differentiation.

Articular cartilage tissue defects cannot be repaired by the proliferation of resident chondrocytes. Autologous chondrocyte transplantation (ACT) is a relatively new therapeutic approach to cover full thickness articular cartilage defects by in vitro grown chondrocytes from the joint of a patient. Therefore, we investigated the redifferentiation capability of human chondrocytes maintained in alginate culture. The cartilage specimens obtained from 50 patients who underwent total knee and hip operations at the teaching hospital of Isfahan University of Medical Sciences, Isfahan Iran. Isolated primary chondrocytes were first grown in monolayer cultures for 1 to 6 passages (each passage lasting about 3 days). At each passage, monolayer cells seeded in alginate culture and investigated morphologically and immuno-cytologically for expression of cartilage-specific markers (collagen type II and cartilage-specific proteoglycans). The chondrocytes from monolayer passages P1 to P4 introduced in alginate cultures regained a chondrocyte phenotype. Cells were interconnected by typical gap junctions and after few days, they produced a cartilage-specific extracellular matrix (collagen type II and cartilage-specific proteoglycans). In contrast, cells from monolayer passages P5 and P6 did not redifferentiate to chondrocytes in the alginate cultures. Chondrocyte culture was established for the first time in Iran. The alginate culture conditions promote the redifferentiation of dedifferentiated chondrocytes that have still a chondrogenic potential. This procedure opens up a promising approach to produce sufficient numbers of differentiated chondrocytes for ACT. Indeed, in some patients the harvested cells were used immediately and successfully for transplantation.

In order to provide a pharmacological profile for some newly synthesized dihydropyridines, we investigated their effects on the isolated rat colon segments and the isolated rat atrium contractility. The tested compounds include alkyl ester analogues of nifedipine, in which the ortho-nitrophenyl group at position 4 is replaced by 2-alkylthio-1-benzyl-5-imidazolyl substituent, and nifedipine as a positive control substance. Isolated rat colon and atrial tissues were prepared. Rat colon was contracted with 80 mM KCl, and maximum response was recorded (100%). After washing tissue with Krebs solution it was preincubated with different concentrations of test compounds and again KCl was added and percent change in contraction was calculated. Spontaneous contractions and its frequency for colon and atrium before and after addition of test compounds were also recorded and percent change was calculated. Nifedipine (10-8 -10-5 M) was used as positive control at all experiments.The compounds showed similar effects to that of nifedipine on the isolated rat colon. The potency of these analogues with concentration range 10-5 to 10-4 M was compared to potency of nifedipine which was effective at 10-8 to 10-5 M (P<0.01). However, unlike nifedipine, the test compounds exerted significant positive inotropic effect on the isolated rat atrium (P < 0.01). Our observations suggest that these analogues of nifedipine selectively enhance contractility of heart muscle while causing relaxation of intestinal smooth muscle. These compounds may serve as valuable probes to develop novel dihydropyridines with dual smooth muscle relaxant effect and positive inotropic action.

Phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to yield Pi and diacylglycerol. Two different forms of PAP in rat hepatocyte have been reported. PAP1 is located in cytosolic and microsomal fractions and participates in the synthesis of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine, whereas the other form of phosphatidate phosphohydrolase (PAP2) is primarily involved in lipid signaling pathways. In rat liver,PAP2 has two isoforms; one PAP2a and another PAP2b. In this study, essentialhistidine residues were investigatedin native form of rat purified PAP2b withdiethylpyrocarbonate.PAP2b purified from rat liver plasma membrane by solubilizing with n-octyle glucoside and several chromatography steps. Gel electrophoresis (SDS-PAGE) performed on purified enzyme in order to evaluate its purity and to measure the molecular weight of the enzyme subunit. The enzyme inactivated with diethylpyrocarbonate (DEPC) and the number of moles of histidine residues modified per mol of enzyme determined. The specific activity of purified enzyme was 7350mU/mg protein and it showed only a single band on SDS-PAGE with a MW of about 33.8 kDa. The PAP2b inactivated by DEPC. The maximum 6 moles of histidine residues modified per mole of PAP2b, when about 90% of enzyme activity is lost with DEPC.The data showed that the incubation of PAP2b by DEPC can inhibit enzyme activity. Our findings also, revealed the presence of essential histidines in the structure of PAP2b which involve in its activity. This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate.

Resistance to antimicrobial agents, particularly metronidazole and clarithromycin, is frequently observed in Helicobacter pylori and may be associated with treatment failure. This resistance rate varies according to the population studied. The aim of this study was to assess the pattern of antimicrobial resistance of H. pylori isolates from dyspeptic patients in Isfahan. Antral gastric biopsies from 230 dyspeptic patients were cultured. Susceptibility testing to commonly used antibiotics performed on pure cultures of 80 H. pylori-positive isolates by Modified Disk Diffusion Method (MDDM). Genomic DNA extracted and subjected for study of entire genomic pattern using Random Amplified Polymorphic DNA- Polymerase Chain Reaction (RAPD-PCR). The overall rates of primary resistance were 30.0%, 8.75%, 6.25%, 3.75%, 3.75%, and 2.50% for metronidazole, ciprofloxacin, clarithromycin, azithromycin, tetracycline, and amoxicillin, respectively. Multiple antibiotic resistances were observed in 8 of 27 resistant isolates (29.6%) that mainly were double resistance with the prevalence of 6.25%. No association between antimicrobial resistance and either the gender, age or clinical presentation of the patients were detected. In RAPD-PCR, great diversity observed in 27 resistant strains isolated from different patients and this heterogeneity was not significantly different from susceptible strains. Primary H. pylori resistance to metronidazole in our population was lower than the developing world and even other parts of Iran, to ciprofloxacin was considerable in comparison with results in most other countries. Moreover, antibiotic resistance had no effect on genomic pattern of H. pylori isolates. Finally, pretreatment H. pylori isolates susceptibility testing is highly recommended.

The main objective of this study was to investigate the status of chromosome stability in 3 human-mouse hybridoma cell lines over a period of time in various passages.Metaphase spreads from 3 human-mouse cell lines (HF2X653, SPMO-4 and F3B6) that had been cultured in 4 successive passages, from 1 to 4 weeks, were prepared and analyzed. Metaphase chromosomes stained in Giemsa and a fluorescent dye, Hoechst 33258, for differential staining. This staining was performed for differentiating human and mouse chromosomes.Numerical chromosome analysis showed that although in successive passages the total number of chromosomes in hybridoma cells remained unchanged, some changes occurred in the number of human and mouse metacentric and acrocentric chromosomes during different passages. These changes were detectable, using fluorescence staining method. Since one of the main uses of human-mouse hybridoma cells is producing monoclonal antibody, chromosomal instability in these cells causes the loss of human chromosomes coding the antibody of interest occasionally. Therefore, cytogenetical analysis and characterization of these cells, especially by using the appropriate ways of chromosomal identification, is essential prior to use.

Previous studies have demonstrated that the nitric oxide (NO) dependent death of murine peritoneal macrophages activated in vitro with IFN-g and LPS is mediated through apoptosis. In the present study, we investigated the synergistic effect of LPS, IFN-g and iron on NO production and apoptosis.After determination of iron cytotoxicity, the peritoneal macrophages of Balb/c mice were cultured with iron, LPS, and IFN-gseparately, or a mixture of these for 18 hr at 37 ◦C. Then after 18 hr incubation, the level of NO in supernatant was measured by the Griess method. At the same time, after incubation with ethidium bromide and acridine orange dye, the apoptotic macrophages were detected by fluorescence microscopy. NO production was significantly greater than the control group in macrophages exposed to iron,LPS, or IFN-g alone (P=0.02), while no significant difference was detected in apoptosis rate in the presence of LPS (P=0.08). However, the differences were remarkable between NO production and apoptosis rate in the presence of iron, LPS and IFN-g (P≤0.05).These findings indicate the immunostimulatory effect of iron on NO production by IFN-g and LPS.