Cell Journal (Yakhteh)
Volume:12 Issue: 1, 2010

Helicobacter pylori (H. pylori) cytotoxin and its heterogeneity amongst strains has been closely linked to the varying infection-associated clinical outcomes. In order to determine the decisive role of the vacuolating cytotoxin (vacA) gene mosaicism in its corresponding gene expression and phenotype, we aimed to characterize vacA alleles of different H. pylori strains in addition to the resulting protein and its vacuolating activity in epithelial cell culture. vacA gene polymorphism was determined for 80 H. pylori strains isolated from dyspeptic patients, using multiplex gene-specific polymerase chain reaction (PCR). VacA protein was detected by immuno-blotting assay using a polyclonal anti-VacA antibody. In vitro cytotoxicity assay was conducted on HeLa cells in order to evaluate the vacuolating cytotoxin activity. Genotyping revealed the following strain distribution: 26 (32.5%) s1m1, 35 (43.8%) s1m2, and 19 (23.8%) s2m2 subtypes. Infection with s1m1 type strain was significantly associated with gastric cancer as compared to non-ulcer dyspepsia (p=0.005) and peptic ulcer disease (p=0.008). A 95-kDa immuno-reactive band that represented the vacuolating toxin was demonstrated in SDS-PAGE analysis of concentrated culture filtrate (CCF) of H. pylori strains. H. pylori CCFs induced HeLa cell vacuolation which correlated with the strain genotype; s1m1 strains demonstrated higher levels of vacuolation as compared to s1m2 strains, whereas s2m2 strains showed no detectable cytotoxic activity. The current study confirmed the relatively high cytotoxic activity of s1m1 type H. pylori strains which infect the majority of patients suffering from gastric cancer and may be partly responsible for the pathogenesis of this mortal disease.

Previous studies have shown that transplantation of bone marrow stromal cells (BMSCs) into the contused spinal cord improves functional recovery and that administration of Schwann cells (SCs) after spinal cord injury (SCI) facilitates axonal regeneration. Although the efficacy of these treatments have been proven, when used individually, their resulting number of regenerated axons is small and locomotor recovery is modest; therefore, we decided to research whether co-transplantation of these cells can improve the outcome. Adult male Wistar rats (n=56), each weighting 250-300 grams were used. BMSCs and SCs were cultured and prelabeled with BrdU and 1,1' dioctadecyl 3,3,3',3' tetramethylindocarbocyanin perchlorate respectively. Contusion model of SCI was performed at the T8-9 level using NYU device (New York University device). The rats were divided into seven groups, each consisting of 8 animals. These groups included: a control group, three experimental groups and three sham groups. In the control group, only a laminectomy was performed. The three experiment groups were the BMSC, SC and co-transplant groups, and 7 days after injury, they received intraspinal BMSCs, SCs and the combination of BMSCs & SCs respectively. The sham groups received serum in the same manner. Locomotion in the groups was assessed using the basso, beatie and bresnahan (BBB) test at 1, 7, 14, 21, 28, 35, 42, 49 and 56 days after SCI. More significant improvement was observed in the BBB scores of the co-transplant group (p<0.05) in comparison with BMSC and SC groups. This study shows that co-transplantation of BMSCs and SCs may provide a powerful therapy for SCI and become required for the development of combinatory treatment strategies in the future.

This study was carried out to investigate the oncolytic effect of the Newcastle disease virus (NDV) strain AF2240 on the MCF-7 breast cancer cell line. The NDV-AF2240 was propagated in 11 days old embryonated chicken eggs for 72 hours. The virus in the allantoic fluid was harvested and purified. The haemagglutination (HA) test was conducted on the purified virus to determine the virus titre which was 16384 haemagglutination units (HAUs). The microculture tetrazolium assay (MTA) was carried out via two methods-the monolayer and co-culture techniques- to determine the inhibitory concentration (IC50) of NDV-AF2240 against the MCF-7 breast cancer cell line. Confocal laser scanning microscopy was carried out on polyclonal chicken antibody and fluorescein isothiocynate (FITC) conjugated goat antichicken antibody to observe virus localization in the cells. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was conducted to quantify the percentage of apoptotic cells. IC50 value of NDV-AF2240 was two HAUs in both the monolayer and co-cultures. Virus particles were detected in the cytoplasm of MCF-7 breast cancer cell line after 24 and 48 hours post treatment. Virus budding was detected 72 hours post treatment. The number of apoptotic cells was significantly increased (p<0.05) 72 hours post NDV-AF2240 treatment. The findings of this study show that NDV-AF2240 has an oncolytic effect against the MCF-7 breast cancer cell line. Further studies are needed to understand the anti cancer mechanism of this virus.

The polymorphic variants at codon 72 of the p53 gene, encoding proline or arginine at residue 72, produce marked changes in the p53 structure. From the evidence that the DNA mismatch repair system and p53 interact to maintain genomic integrity, we hypothesized that codon 72 variations may influence the prevalence of microsatellite instability (MSI), a feature of malignancies associated with mismatch repair deficiency in sporadic colorectal cancer. We investigated the frequency of MSI in three P53 codon 72 genotypes using genomic DNAs from 144 paraffin blocks of sporadic colorectal adenocarcinomas by testing the BAT-26 poly(A) marker. We used PCR-SSCP analysis to detect tumor sample MSI for the nonisotopic detection of deletions in the BAT- 26 poly (A) mononucleotide repeat. Associations between qualitative variables were evaluated using the χ2-test. Statistical significance level was set to p ≤ 0.05. MSI analysis revealed that 24.3% of the tumors (n=35) were MSI-positive and 75.7% (n=109) were MSI-negative. The frequency of microsatellite instability in the arginine/arginine, arginine/proline and proline/proline genotypes were 11 (16.9%), 22 (36.1%) and 2 (11.1%) respectively. A significant difference in distribution of MSI was found for the arginine/proline genotype compared with the grouped arginine/arginine and proline/proline genotypes (p=0.05). Our findings suggested that colorectal adenocarcinomas arising in individuals with the p53 codon 72 arginine/proline heterozygosity are more prone to microsatellite instability than those with other p53 genotypes. In our study, MSI was important in the carcinogenesis of sporadic colorectal cancer arising in pro/arg heterozygotes.

This study was performed to determine the effects of solder fume on liver serum markers and vascular elements in rats A total number of 48 rats were randomly divided into experimental (n=30) and control (n=18) groups. Based on exposure time, each group was further divided into 2, 4 and 6 weeks of exposure subgroups. Rats in the experimental subgroups were placed in an exposure chamber and exposed to solder fume for one hour per day. The concentration of metal fumes and gases in the exposure chamber were measured daily by atomic absorption spectrophotometry. Blood and liver samples were collected from all rats in the experimental and control subgroups to determine the concentration of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, as well as total and conjugated bilirubin levels. Histological examinations of specimens were performed under a stage-micrometer calibrated microscope. Despite some alterations in enzyme and bilirubin levels between control and experimental groups, the differences were not statistically significant. However, histopathological examinations of liver tissues revealed significant differences in the diameters of cross sectioned sinusoids of rats in the 4-week experimental and control subgroups (p<0.001). Furthermore, there were significant differences in the diameters of cross-sectioned central venules of all experimental and control subgroup members (p<0.001). The results of this study suggest that although solder fume in the acute phase of exposure of up to 4 weeks does not significantly change liver serum marker enzymes, it can greatly increase the diameters of cross-sectioned hepatic sinusoids and the central venule.

Diazinon (DZN) is an organophosphorate synthetic insecticide widely used in agriculture. DZN has been observed to cause many changes, such as alterations in androgenic hormones. In the present study, the effect of DZN treatment on the structure of the testes and spermatogenesis in young adult Albino rats was evaluated. Adult rats were randomly divided into three groups including: controls (n=6), DZN-treatment group A (n=6) and DZN-treatment group B (n=6). Commercial DZN was injected intraperitoneally in a single dose (A=25 mg/kg and B=2.5mg/ kg), corresponding to LD50. Thirty five days after injection, animals were sacrificed for morphological and histological examinations. There was a significant reduction in seminiferous tubule size in group A in comparison with both controls and group B (p<0.001). The number of spermatocytes, Leydig and germinal cells were significantly decreased (p<0.001). These differences were not significant between the controls and group B; however, the number of spermatocytes in group B was significantly lower than in the controls (p<0.01). This study revealed that the reproductive function of adult rats and spermatogenesis are sensitive to DZN treatment. In addition, the effect of DZN on morphological parameters was significantly dose dependent. Further study of the control DZN and the actual mechanism whereby it exerts toxic effects on male infertility is required.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by congenital malformations, bone marrow failure, development of squamous cell carcinoma (SCC), and other cancers. Human papillomavirus (HPV) in the oral cavity or oropharynx has been associated with an increased risk of laryngeal papillomatosis, invasive squamous cell carcinoma of the head and neck (HNSCC) and cervical and other genital cancers. The prevalence of HPV DNA in the oral cavity/oropharynx in FA patients and controls was compared. A risk factor questionnaire and oral exfoliated cells were collected from FA patients. The study group consisted of 22 FA patients with HNSCC (case subjects) and 24 patients with HNSCC without FA (control subjects). HPV DNA was detected using polymerase chain reaction (PCR) and specific primers that covered high risk types of HPV. Moreover, special serological assays were used for the detection of specific antibodies against HPV in patient’s sera. HPV DNA was detected in 82% of the SCC specimens from the case subjects which was statistically higher (p< 0.05) than the SCC specimens from the control subjects (62.5%). In all cases, the presence of HPV antibodies in patient’s sera has been shown. Among the case subject specimens, 14 cases, and in the control subjects, 11 cases were infected by high risk HPV (hr-HPV). These data confirm that HPV infection, especially with high risk types (16,18), could be one of several risk factors for HNSCC particularly in FA patients.

Transplants of multipotent stem cells have been shown to have a neuroprotective effect after central nervous system injury. The bulge region of the hair follicle has been reported as a putative source of hair follicle stem cells (HFSC) for many years; however, few studies have documented the properties of bulge derived cells in vitro until now. This study was conducted to isolate and culture bulge cells from rat hair follicles and to determine the morphological and biological features of the cultured cells. The bulge region of the rat whisker was isolated and cultured in Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) supplemented with epidermal growth factor (EGF), cholera toxin. Dissociated bulge stem cells were differentiated on coated substrates together with NT-3. The morphological and biological features of cultured bulge cells were observed by light microscopy and immunocytochemistry methods. Our results showed that newly proliferated cells could be observed on the 4th day after explantation. The expression of a neural progenitor marker, nestin, was seen before differentiation of the bulge cells. The differentiated cells expressed βIII-Tubulin and RIP, which are the markers of neural and glial lineages. The results indicated that the bulge cells cultured from the rat hair follicle had the characteristics of stem cells and could differentiate into neural and glial lineages.

Animals of the inbred BDII rat strain are genetically predisposed to endometrial adenocarcinomas (EAC) and can be used to model human cancer. From our previous studies, it was obvious that some chromosomes were selectively involved in EAC development; one of them was rat chromosome (RNO) 15, in which there were often losses in the short arm and gains in the long arm. Since cytogenetic events lead to allelic imbalance and/or loss of heterozygosity (AI/LoH) in RNO15, it was subjected to a detailed analysis with polymorphic microsatellite markers spanning the entire chromosome. BDII/Han females were crossed to males from two other inbred rat strains known to have low incidence of EAC (BN/Han and SPRD-Cu3/Han). DNA extracted from F1, F2 and backcross offspring were used in this studies. Our final marker panel consisted of 36 markers. The analysis showed that AI/LoH was common in EAC tumors and was concentrated to four well-defined regions along the chromosome. Two of these regions were close to the distal end of the short arm; one region was in the middle of the chromosome, probably spanning the centromere; and the fourth region was located distally in the long arm. According to the Rat Genome Project (RGP), the number of genes in these regions approached 300. According to a database search, about 80 of these genes could be considered “cancer-related” and they were potential candidates to be targets for the observed chromosomal aberrations. Among the cancer-related genes, there were Anxa7 (Region I), Bmp4, Lgals3, Cdkn3 (Region II), Rb1, Ddx26, Clu, Bnip3, Nkx3.1 (Region III), and Gpc5 (Region IV).

Mesenchymal stem cells (MSCs) can be expanded and differentiated into many mature cell types including smooth muscle cells (SMCs). In addition to growth factor, cyclic stretch contributes to differentiation of stem cells. Mechanical stimuli are critical to morphological changes, development, regeneration, differentiation and pathology of mesenchymal tissues. The aim of this study is to investigate effects of cyclic stretch with differing amplitudes on morphology and differentiation of mesenchymal stem cells. Mesenchymal stem cells are extracted from human bone marrow. Cells are cultured on silicone membrane and exposed to cyclic stretch by a custom made device. Cellular images are captured before and after tests. Effects of 5% and 15% uniaxial strain with 1Hz frequency and 1-8 hour durations on morphology of human mesenchymal stem cells are investigated. It is assumed that environmental factors such as mechanical loading regulate MSCs differentiation to SMCs. Fractal analysis is used to quantify alterations in cellular morphology. An image processing method with a designed code is used for evaluation of fractal dimension parameter. Results demonstrate statistically significant change in cell morphology due to mechanical stretch. By elevation of strain amplitude and number of load cycles, fractal dimensions of cell images decrease. Such decrease is equivalent to alignment of cells by mechanical stimulus. Cells are differentiated to SMCs purely by cyclic stretch. The initiation and rate of differentiation depend on mechanical conditions. To produce functional SMCs for engineered tissues, MSCs can be exposed to uniaxial cyclic stretch. The functionality of differentiated SMCs depends on loading conditions.

Development of the central nervous system (CNS) is dependent on interactions between genetic and epigenetic factors, some of which could affect the susceptibility of the developing brain to damaging insults. Gestational stress has been shown as a potential factor associated with higher risk of developing certain neurological and psychiatric disorders. This study tested the hypothesis that maternal stress influences the risk of epilepsy in offsprings. Pregnant mice were exposed to restraint stress twice a day for three days at the start of the last week of gestation. Ten days after birth, the intact hippocampi of the newborn mice were excised and prepared for investigation. The hippocampi were bathed in low magnesium artificial cerebrospinal fluid to induce field potential, and the subsequent spontaneous seizure-like events of the CA1 neurons were recorded. Plasma corticosterone was measured using a commercial radioimmunoassay (RIA) kit and the values were expressed as μg/100 ml. Both the number of recurrent seizures and the duration of seizure activity were reduced in the stressed group compared to the controls (p<0.001). Stress induced a significant rise in serum corticosterone levels in both pregnant mice and in their newborn pups (p<0.001). These findings suggest that acute prenatal stress, which may mimic acute stress in human pregnancy, is a likely factor affecting seizure control in childhood temporal lobe epilepsy. The underlying inhibitory mechanism may be an increase in the level of neurosteroids both in the blood and the brain.

This study was designed to examine the effects of human umbilical cord matrix stem cell (hUCMSC) administration in rats for 6 weeks after traumatic brain injury (TBI). Adult male Wistar rats (n = 30) were injured with controlled cortical impact device and divided into three groups. The treatment group (n = 10) was injected with 2 × 106 hUCMSC intravenously, the vehicle group (n=10) received phosphate buffered saline (PBS) whereas the control group (n = 10) receive nothing. All injections were performed one day after injury into the tail veins of the rats. All cells were labelled with Brdu before injection. Evaluation of the neurological function of the rats was performed before and after injury using Neurological Severity Scores (NSS). The rats were sacrificed 6 weeks after TBI and brain sections were stained using Brdu immunohistochemistry. Statistically significant improvement in functional outcome was observed in the treatment group compared with the control group (p < 0.01). This benefit was visible 1 week after TBI and persisted for six weeks (end of trial). Histological analysis showed that hUCMSC were present in the lesion boundary zone at 6 weeks in all cell injected animals. Rats injected with hUCMSC after TBI survive for at least six weeks and show functional improvemnt.

The aim of this study was to clone PPARγ1 cDNA in an appropriate mammalian expression vector, with a chimeric cDNA form, encompassing PPARγ with enhanced green fluorescent protein (EGFP) cDNA. This recombinant plasmid will be used for further analyses to investigate the molecular mechanism of PPARγ1 for neural differentiation process. Moreover, the nuclear localization of the PPARγ1 protein linked to EGFP marker was chased by using transient transfection of a constructed plasmid into bovine fibroblast cells. Total RNA was extracted from the fatty tissue of an adult mouse. Using specific pair primers, PPARγ1 cDNA was synthesized and amplified to produce the entire length of ORF. RT-PCR products containing PPARγ1 cDNA were treated by enzymatic digestion and inserted into the pEGFP-C1 downstream from EGFP cDNA. The constructed vector was used for transformation into bacterial competent cells. Positive colonies which showed inserted PPARγ1 cDNA were selected for plasmid preparations and additional analysis was performed to ensure that PPARγ1 cDNA was inserted properly. Finally, to confirm the intracellular localization of EGFP-PPARγ1, bovine fibroblast cells were transfected with the recombinant plasmid. Our results from enzymatic digestion and sequencing confirmed, as expected, that PPARγ1 cDNA was amplified and cloned correctly. This cDNA gene encompassed 1428 bp. The related product was entered into the nucleus of bovine fibroblasts after transfection of its cDNA. PPARγ1 cDNA was cloned and sorted into nuclear compartments of bovine fibroblast cells upon transfection.

The purpose of this study was to investigate the enhanced thermal resistance mechanism of the DU145 tumor spheroid cultures as compared to the prostate carcinoma cell line's monolayer cultures. DU145 cells were cultured either as spheroids or monolayers. Cultures were treated with hyperthermia in a precision water bath (at 43°C for 60 minutes) and/or quercetin (50 and 500 μM for monolayer and spheroid cultures respectively). After hyperthermic treatment, the cell viability colony forming ability, and the expression of heat shock protein 70 (Hsp70) were examined in both culture systems. Hsp70 expression was studied using the western blot method. Our results showed that the DU145 monolayer and spheroid cell culture treatment with hyperthermia alone resulted in a marked survival inhibition. Furthermore, the spheroids showed a more significant resistance to hyperthermia compared to the monolayer cultures (p = 0.01). They also produced more Hsp70 than the monolayer cultures. Treatment of cells with quercetin reduced the Hsp70 level in both culture systems. However, with the reduced Hsp70 levels, thermal resistance of the spheroids showed a greater decrease in relation to that of the monolayers. The results suggest that the enhanced hyperthermia resistance mechanism of the spheroid cultures compared to that of the monolayer cultures can be attributed to spheroid's Hsp70 production.

The aim of this study was to investigate the effects of two vitrification protocols on mouse metaphase stage II (MII) oocytes and evaluate their effects on the expression of heat shock protein A1A (HSP A1A) and MnSOD genes.

Groups of approximately 15 oocytes without cumulus complexes were collected and vitrified with 10% (v/v) ethylene glycol (EG) + 10% (v/v) dimethylsulphoxide (DMSO) + 0.5M sucrose in group A (VSI) and 14.5% (v/v) EG + 14.5% PROH + 0.5M sucrose in group B (VSII), respectively. Thawing after vitrification was performd by placing the oocytes into 1M sucrose for 1 minute and two diluted solutions, each for 3 minutes. After thawing, the oocytes were fertilized and cultured in vitro to develop into the pronuclear stage. The survival rate of vitrified-warmed oocytes and rate of fertilization were evaluated. In addition, gene expressions (HSP A1A, MnSOD and β actin) of vitrified-warmed oocytes were also examined by reverse transcription polymerase chain reaction (RT-PCR).

Survival rates of each group were separately compared to the control. The result showed significant differences between each experimental group compared to the control (p≤ 0.001). The survival rate of oocytes after warming was higher in VSI (91.2% ± 1.7) compared to VSII (89.2% ± 1.5) but there were no significant differences between the two groups. The rate of fertilization was significantly (p≤0.05) reduced in vitrified-warmed (VSI: 39% ± 5.8; VSII: 34% ± 5.7) oocytes compared to the control (88.36% ± 2.3). The expression of MnSOD increased in the vitrified-warmed oocytes when compared to control oocytes. We also detected HSP A1A only in the control and VSI group.

Vitrification of oocytes by cryotop resulted in high survival rate; low developmental competence and fertilization rate of vitrified-warmed oocytes which may be a result of changing expression of important genes after thawing.