Iranian Biomedical Journal
Volume:11 Issue: 1, Jan 2007

The p53 protein function is essential for the maintenance of the nontumorigenic cell phenotype. Pancreatic tumor cells show a very high frequency of p53 mutation. To determine if restoration of wild type p53 function can be used to eliminate the tumorigenic phenotype in these cells, pancreatic tumor cell lines, PANC-1 and HTB80, differing in p53 status were stably transfected with exogenous wild type p53 gene. The transfection was performed using Polybrene/DMSO-Assisted Gene Transfer method. The wild type p53 gene integration into genomic DNA was detected by Southern blot and PCR. Furthermore, the expression of wild type p53 protein was detected in selected clones by immunohistochemistry and Western blot. While HTB80 cell line failed to produce a stable p53 expressing clone, the PANC-1 cells produced stable lines. Following characterization of clones, the growth rate and tumorigenicity of PANC-1 wild type p53 clones were compared to the control cells. Our data showed that the expression of wild type p53 decreased the growth rate of PANC-1 cells. It was also observed that the expression of wild type p53 in PANC-1 cells suppressed its potential for tumor formation in nude mice, completely, while the parental line leads to the formation of a relatively large tumor. Our results suggest that gene therapy based on restoration of wild type p53 protein function in pancreatic tumor cells with high amount of mutant p53 is a feasible option in pancreatic cancer treatment. Iran. Biomed.

High levels of rheumatoid factors (RF) are detectable in serum of the majority of patients with rheumatoid arthritis (RA), but 5-10% of patients remain seronegative (SN). Despite clinical and genetic similarities between these two subsets of RA, it has been proposed that they may be regarded as distinct clinical entities. In the present study a panel of monoclonal antibodies (mAb) recognizing RF-associated cross-reactive idiotypes (CRI) linked to the VH1 (G8), VH4 (LC1), VK3b (17-109) and a mAb recognizing the VK3 subgroup (C7) of immunoglobulin variable region (IgV) gene products were used to quantitate the level of expression of these gene products in serum and synovial fluid of 35 seropositive (SP) and 8 SN RA patients by capture ELISA. While the concentration and relative proportion of the IgV are recognized by the mAb G8, 17-109 and C7 were significantly higher in serum and synovial fluid of the SP RA, compared to the SN-RA patients (G8, p = 0.009; 17-109, p = 0.0001; C7, p = 0.001). The CRI recognized by the mAb LC1 was highly represented in serum and synovial fluid of the SN-RA patients. There have been no significant differences in the level of expression of these IgV gene products (other than the product recognized by C7 mAb in SP patients) between serum and synovial fluid of either group of patients. Our results suggest that the expressed repertoire of Ig VH and VK genes in these two subsets of RA is differentially regulated and may be influenced by selective mechanisms leading to positive or negative selection of certain genes. Iran. Biomed.

Busulfan, a cytotoxic drug, which is currently used as a chemotherapeutic agent, has many side effects on different body organs. In this research, the effects of busulfan on sperm parameters and microstructure of mouse testis were investigated. Busulfan was injected intrapretoneally at 10, 20, 30, 40 and 50 mg/kg and testes were removed after 4, 6 and 8 weeks, weighed and processed for light microscopic examination. Transverse and cross section diameters of testes, seminiferous tubules diameters, percentage of different types of tubules, epithelium thickness, spermatogenic cell numbers and capsule thickness as well as the sperm parameters in epididymis were measured. There was a significant decline in sperm numbers and marked changes in testes structures. Almost 8 weeks after the injection of drug, some of the changes are reversed. Accordingly, the changes in percent of normal tubules without sperm, abnormal tubules and capsule thickness were increased until 6 weeks of drug administration, the changes declined thereafter. In general, busulfan caused a decrease in all analyzed parameters (except capsule thickness, normal tubules without sperm and abnormal tubules), probably due to the arrest of spermatogenesis. Our results also revealed that some of the changes are reversible and dose dependent. Iran. Biomed.

CD8+ T cells are thought to play an important role in protective immunity to tuberculosis. The major histocompatibility complex class I subtype HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A*0201 transgenic, H-2Db/mouse beta2-microglobulin double-knockout mice (HHD) which express human HLA-A*0201 but no mouse class I, was shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. HHD mice were immunized with plasmid DNA encoding MPB51 by using a gene gun, and IFN-g production from the immune spleen cells was analyzed in response to a synthetic overlapping peptide library covering the mature MPB51 sequence. catatonic T lymphocytes (CTL) activity was measured using cytotoxicity assay and the three-color flowcytometry was used to reveal IFN-g-producing immune spleen cells. Our findings were shown that only one peptide, p51-70, appeared to stimulate the immune splenocytes to produce IFN-g. Flow cytometric analysis with intracellular IFN-g and the T-cell phenotype revealed that the p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis with computer-assisted algorithms permitted identification of a T-cell nona mer epitope, p54-62. Finally, we proved that the p54-62/HLA-A*0201 complex is strongly recognized by HLA class I-restricted CD8+ MPB51-specific CTL cells. These results suggest that vaccination with MPB51 gene elicited MPB51-specific CTL. In addition, the P54-62 epitope thus represent potential subunit component for the design of vaccines against tuberculosis. Iran. Biomed.

Serotonin is believed as an important factor in brain function. The role of serotonin in cerebral psycho-patho-physiology has already been well established. However, the function of serotonin antagonist in anesthetized subjects under hyperthermia has not been studied properly. Experiments were performed in three groups of urethane-anesthetized rats, such as: (i) control group, (ii) whole body hyperthermia group and (iii) p-CPA (para-Chlorophenylalanine) pretreated hyperthermia group. Hyperthermia was produced by subjecting the rats to high ambient temperature of 38 ± 1ºC (relative humidity 45-50%). Each group was divided for EEG (electroencephalogram) study and for determination of edematous swelling in the brain. Urethane anesthetized rats under hyperthermia show highly significant reduction in their survival time. The body temperature recorded during the hyperthermia was observed with significant and linear rise with marked increase in brain water content, which was analyzed just after the death of the subjects. The results of the electroencephalographic study in urethane-anesthetized rats recorded before death indicate that brain function varies in systematic manner during hyperthermia as sequential changes in EEG patterns were observed. However, a serotonin antagonist, p-CPA pretreatment increases the survival time with significant reduction in edematous swelling in brain but it does not affect the relationship between the core body temperature and the brain cortical potentials as observed in urethane anesthetized subjects exposed to whole body hyperthermia. The core body temperature in p-CPA pretreated rats show non-linear relationship with respect to the exposure time as it was observed in drug untreated subjects. The findings of the present study indicate that although pretreatment of p-CPA in rats has a marked correlation between the extravasations of the blood-brain barrier under hyperthermia but shows minimum effect on the EEG in a model of hyperthermia under irreversible anesthesia. Iran. Biomed

Neuroserpin, a member of the Serine Proteinase Inhibitor (Serpin) superfamily, is known to be a neuroprotective factor in the focal ischemic stroke followed by reducing the microglial activation. Neuroserpin is a protein rich of methionine residues that can scavenge the free radical species which may increase its neuroprotective effect. On the other hand, the oxidative modifications of the amino acid residues in neuroserpin may lead to changes in its conformation and function. In this study, it was investigated the changes in the conformation and the function of the oxidized neuroserpin. Neuroserpin expressed in E. coli, BL21 or M15 harboring plasmid pQE81L containing neuroserpin cDNA. Expressed neuroserpin was purified by resin sulfopropyl A50 precharged with 0.1 M NiSO4 under denaturing condition. Neuroserpin was oxidized under oxidative stress condition in the presence of different concentration of hydrogen peroxide. The oxidation of neuroserpin was conveniently detected by a carbonyl content assay using 2, 4 dinitrophenylhydrazine. Changes in tertiary structure of neuroserpin were monitored by spectrofluorimeter to study the alteration of intrinsic fluorescence and also fluorescence of 8-anilinonaphthalin-1 sulfonic acid (ANS) in native and oxidized form of neuroserpin. Total expressed neuroserpin was estimated 4-5 mg/lit in 2XYT culture media. SDS-PAGE analysis of purified neuroserpin showed a single band which reflects the efficiency of the resin SP A50 for purification of the proteins containing 6×His tag. Carbonyl content of oxidized and native neuroserpin was estimated 12.3 ± 0.3 and 0.45 ± 0.05, respectively. The inhibitory activity of oxidized neuroserpin decreased up to 40-60% as compared with native form of neuroserpin. Intrinsic fluorescence and also the emission of ANS bind to the hydrophobic region of the protein altered from 380 to 85 and in the case of ANS from 105 to 150 in oxidized and native form of neuroserpin, respectively. The decreased intrinsic fluorescence intensity, an enhancement in the fluorescence of ANS, and loss of the inhibitory activity up to 40-60% in neuroserpin, all suggested a conformational modification in the protein under the oxidative stress condition. Remaining the inhibitory activity of neuroserpin reflects that the protein tolerates the oxidative stress condition effectively. Iran. Biomed.

The aim of this study was to compare invasive and non-invasive strains of Shigella flexneri isolated from Tehran by a 120 kDa protein band by SDS-PAGE, electron microscopy of cell culture and Congo red dye methods. S. flexneri strains were isolated by standard bacterial methods from fecal specimens of children attending to the 3 children’s hospitals. Phenotype analysis for screening virulent of strains of S. flexneri was done on a plate of tryptic soy agar contained 0.003% Congo red dye. Whole membrane protein preparations were used to examine the protein profiles of the inner and outer membrane of these Gram-negative bacteria. The protein mixture was electrophoresed through a polyacrylamide gel. The gel was stained with Coomassie brilliant blue R250 and destained with ethanol and acetic acid. HeLa cell culture was done by two-step preparations: one for light microscopy and the other for electron microscopy. Some of S. flexneri (46%) were Congo red positive colonies. S. flexneri with negative Congo red phenotype could not enter the HeLa cell culture. A 120 kDa protein band was found in 46% of these bacteria which could enter into HeLa cell culture. Pseudopod structures which facilitate bacterial cell-to-cell spread were readily identified by electron microscopy. Since the existence of 120-kDa protein band was corresponded to enter of S. flexneri into the HeLa cell culture and correlated with Congo red dye positive, for identification of invasive and non-invasive S. flexneri strains, the use of a 120-kDa protein band by SDS-PAGE or a simple, rapid and very cheap Congo red dye method is recommended. Because, there are some deaths due to Shigella sp. in our country, notification on the isolation of these bacteria in both children hospitals laboratories and private clinical laboratories is important. Iran. Biomed.

The herpes simplex virus type 1 (HSV-1) tegument protein, VP22 has been reported to have the property of intercellular transport. The previous studies have shown that following expression of a fusion protein containing VP22; it spreads to every cell in a monolayer and concentrates in the nucleus. In spite of these reports, some studies have shown that VP22 trafficking and its nucleus accumulation is an artifact and no improvement in translocation of proteins fused to VP22 has been detected. To better understand about VP22 translocation, VP22-GFP (Green Fluorescent Protein) vector was constructed and its nuclear accumulation, transportation to the nontransfected cells and translocation between different cell types were studied by fluorescent microscope. VP22-fusion protein was detected in nontransfected cells which in some of them the fusion protein was shown in nucleus. The results demonstrated that VP22 can easily transport between different cells but nuclear accumulation of the protein is not common in all of the recipient cells. Iran. Biomed.

The aim of this study was to investigate the prevalence of hepatitis B virus (HBV) genotypes in Isparta, Southwest of Turkey, as well as the clinical features and transmission route for patients with HBV infections. Patients (n = 135) with HBV infection were included in the study. Epidemiological and clinical data were obtained. HBV genotypes were determined with a preS2 epitope ELISA kit. Although the HBV transmission route remained unidentified in 51.1% of the patients, blood contact was determined as the most common probable transmission route (38.5%). One hundred twenty-four (91.8%) of 135 samples, could be genotyped. One hundred fifteen (85.1%) were genotyped as type D/E, six (4.4%) were genotyped as type A, two (1.4%) were genotyped as type C, and one (0.7%) were genotyped as type F. Genotype D/E is determined as the predominant HBV genotype circulating in Isparta, Southwest of Turkey. No relationship between genotypes and disease severity and transmission route has been detected. Iran. Biomed.

Teucrium polium is an analgesic, antidiabetic and antilipeidemic herbal medicament. The aim of this survey was to evaluate the effect of aqueous extract T. polium on liver enzymes linked to liver dysfunction, serum lipids and glucose, in diabetic male rats. A total of 20 Sprague-Dawly male rats became diabetic by intraperitoneal injection of streptozotocin (60 mg/kg). the animals were divided randomly into two groups. Experimental group was fed Teucrium polium (50 mg/kg) for a month but control group was received the same volume of distilled water. Liver enzymes, biochemical parameters (cholesterol, triglyceride, low density lipoprotein, alanine transaminase, aspartae transaminase) and glucose were measured by kinetic (Enzymatic) and colorimetric methods. Data obtained were analyzed and mean values were compared by paired student''s t-test. The results were expressed as mean ± SD. Significant differences were set at P<0.05. Our results showed that in test group, serum glucose values decreased significantly (P<0.05), but cholesterol, triglyceride, low density lipoprotein, alanine transaminase and aspartae transaminase increased significantly after use of T. polium (P<0.05). This parameters value did not show any changes in control group. Although the aqueous extract of Teucrium polium has strong hypoglycemic properties in experimental animals, but because of some hepatotoxic effects, it is not suitable to use it in human as an antidiabetic agent. Iran. Biomed.