Cell Journal (Yakhteh)
Volume:12 Issue: 3, 2010

It has been stated that cells sometimes have the ability to remember who and what they are! They have this ability, even, though they contain although they have all the necessary genes neededwith which to become all typeskinds of cells. In this regard, the pattern of gene expression must be inherited from one cell generation to the next by mechanisms that lie outside the DNA sequence itself, which is termed cellular memory or epigenetic inheritance.Developmental biology is under the control of both genetic and epigenetic mechanisms. Studies show that the regulation of chromatin structure by DNA methylation and histone modification is crucial for genome reprogramming during early embryogenesis and gametogenesis, as well as for tissue-specific gene expression and differentiation. Understanding the process of epigenetic reprogramming in development is important for studies of cloning and the clinical application of stem-cell therapy. In the current review we briefly discuss the molecular mechanism of cellular memory, under the control of epigenetic regulation

In order to achieve the best therapeutic strategy with which to treat multiple myeloma (MM), it is necessary to understand the molecular mechanisms of myeloma cell growth. In addition to genetic defects, the bone marrow microenvironment (BMM) plays a primary role in MM pathophysiology. For many years, interleukin-6 (IL-6) was considered to be a central growth factor in myeloma cells. However, recent evidence indicates that increasing numbers of cytokines enhance myeloma cell growth in BMM. In this study, we investigated the possibility that leptin could be a member of cytokine network that supports myeloma cells.

The expression of leptin and its receptors in cell lines, bone marrow (BM) and mononuclear peripheral blood (PB) were analysed by RT-PCR. Additionally, leptin serum levels were analysed by ELISA and the effect of leptin on growth of cell lines by cell culture.According to our results,leptin and its receptors were expressed in cell lines, myeloma BM and PB mononuclear cells (MNC). However the PB mononuclear cells of treated myeloma patients did not express leptin and differed in the expression of leptin receptors. Acute lymphoid leukemia, B-blasts (B-ALL) did not express leptin and its receptors. Untreated myeloma and B-ALL patients had high and low levels of leptin in their serum, respectively. Recombinant exogenous leptin enhanced the growth of RPMI8866 but did not affect the growth of U66 and Jurkat.Regarding this study and the findings of other studies, leptin may play a role in MM pathophysiology. Therefore leptin and its receptors might be analysed as a therapeutic target for myeloma BMM.

Germline mutations in breast cancer susceptibility genes, breast cancer susceptibility gene (BRCA) and breast cancer susceptibility gene (BRCA) are responsible for a substantial proportion of high-risk breast and breast/ovarian cancer in families. Therefore, the aim of this study was to investigate BRCA/ mutations in five high risk Iranian families. Of the 0 breast/ovarian cancer families counselled in our center, five were selected for BRCA/ mutation screening according to our minimal criteria. The complete coding sequences in addition to each intron/exon boundary of the BRCA/ genes were screened by direct sequencing. Fourteen missense substitutions were identified, which were: Gly40Ser, Gly78Glu, Glu75Glu, leu87pro, Ser6Gly, ser040Asn, Glu08Gly, Leu77Leu and Ser46Ser in BRCA; and Gln7His, Glu9Gly, Leu5Leu, Val7Val and Glu05Glu in BRCA. In addition, the splice site mutations (IVS7+8(-TT) and so IVS8-70 (-CATT) were observed in two families. Three mutations were novel (Gly40Ser in BRCA and Glu9Gly, Gln7His in BRCA). The missense substitutions Glu08Pro and Gly40Ser were found in a large series of patients and in five controls. The missense substitution Gly78Glu in BRCA is pathogenic. In addition, these results showed that the probability genotype at the BRCA locus defined by alleles Leu87Pro, GLu08Gly, Ser6Gly, Gly40Ser has an effect pathogenic. In another family َseveral missense substitutions in BRCA gene such as Glu08Gly, Gly 40Ser were found as well as Glu9Gly and Gln7His in BRCA. The pathogenic effect yet has to be verified by more comprehensive populations studies. These results support this thinking that screening for BRCA and BRCA mutations may have the strongest impact on health-care when targeted to high-risk populations.

Human multiple sclerosis (MS) is a complex disease and demyelinated lesions in central nervous system (CNS) are the pathologic hallmark of MS. Remyelination occurs in many MS lesions but becomes increasingly incomplete/inadequate. Protein tyrosine phosphatase,receptor-type z polypeptid (PTPRZ) has been implicated in adult cell renewal, repair of the nervous system, oligodendrocyte development and so in Remyelination. We investigated possible association of multiple sclerosis with polymorphism of two SNPs (rs478 and rs69657) located in PTPRZ gene.

Peripheral blood was collected from 40 subjects with MS and 65 healthy controls and DNA was extracted. For genotyping of rs478 and rs696575, PCR-RFLP and mismatch PCR-RFLP techniques were used, respectively. Association of SNPrs478 and SNPrs696575 with multiple sclerosis was examined by using the Chi-square test and the frequency differences of alleles and genotypes between two groups were compared. A conventional p-value of ≤ 0.05 was considered significant.

Statistical analyses on two studied polymorphisms showed that both case and control group were in Hardy-Weinberg equilibrium. By using χ test, the difference between frequency of SNPrs478 Risk allele vs. other allele in control and case groups was p=0.77 and for SNPrs696575 was p=0.669. The difference between frequency of Homozygosity vs. other genotypes in control and case groups for SNPrs478 was p=0.77 and for SNPrs696575 was p=0.64.

According to the χ test results, the differences were not significant for studied SNPs. As a conclusion, we did not find association between SNPrs478 and SNPrs696575 of PTPRZ and multiple sclerosis in Iranian population.

The objective of this study was to isolate and culture human dental pulp stem cells to study important stem cell markers in them. Dental stem cells were isolated from human pulp and cultured in alpha-modified eagle’s medium (α-MEM) supplemented with 0% fetal bovine serum (FBS) in a 7°C incubator with 5% CO and photographed under inverted microscope. The expressions of the important stem cell markers were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and agarose gel electrophoresis in the cells at different passages. Cells isolated from dental pulp showed a high rate of proliferation and were cultured to more than 5 passages in vitro. The study of gene expression by RT-PCR showed that these cells expressed nucleostemin, cyclin D, Oct-4 and nanog (major components of the PluriNet) in different passages as well as under serum-free conditions. Cells isolated from dental pulp are genuine pluripotent stem cells with high potential for self-renewal. The expression of the stem cell markers in human dental pulp stem cells indicate that they have a great potential for cell therapy and regenerative medicine, even they were isolated from adult teeth.

Multiple myeloma (MM) is a hematological malignancy characterized by osteolytic bone disease which is associated with severe bone pain and pathological bone fractures. The receptor activator of nuclear factor B (RANK) and receptor activator of nuclear factor B ligand (RANKL) system has an important role in regulation of bone remodeling process. The aim of this study was to evaluate the expression of the RANK/RANKL molecules by the myeloma cells derived from patients and myeloma cell line U-66. Myeloma cells derived from 7 myeloma patients and plasma cell leukemia were included into this study to evaluate the expression of the RANK/RANKL molecules by the reverse transcriptions-polymerase chain reaction (RT-PCR) method at the mRNA level. As well as human myeloma cell line U66, U97, RPMI-8866 and Hela were used as control groups. In this study we show the expression of RANK and its ligand at the mRNA level in U-66 (myeloma cell line) and plasma cells derived from patients by the RT-PCR technique. Our results demonstrate that expression of RANK and RANKL by plasma cells can contribute to induction of osteoclasts and plasma cell activation which elevates bone resorption in myeloma patients.

Yersinia pestis, the causative agent of the zoonotic plague infection, is a major public health concern both as a threat and potential bioweapon. The objective of the present study was to establish a uniplex and multiplex - polymerase chain reaction (PCR) test for the specific detection of Y. pestis. PCR reactions performed by three pair primers which targeted the caf and pla genes located on the pFra and pPst plasmids and the irp chromosomal gene located on the ‘pathogenicity island’. After TA cloning of the PCR products, the test’s limit of detection (LOD) was determined. For evaluating the specificity, PCR reactions were performed with negative control bacteria. Assays were performed with the genome of Y. pestis which produced three DNA fragments of the expected sizes 00, 400 and 50 bp which corresponded to the irp, caf and pla genes, respectively. The lower LoD was 70 copy numbers for the caf gene and for the pla gene. In PCR reactions that used negative control bacteria, detectable fragments were not observed. Our method clearly discriminated Y. pestis DNA. The rapidity, specificity and sensitivity of this procedure suggest that it can serve as a useful alternative method for the inoculation of laboratory animals or the use of specific culture media for routine plaque surveillance and outbreak investigations. Another vital result of this study was the establishment of Y. pestis molecular detection technique in Iran.

Infection with genital Mycoplasmas may have harm effects on the reproductive health of men, thus leading to male infertility. This study was performed to detect the prevalence of these bacteria and to study the sperm parameters in infertile men who referred to Royan Institute during 009. Semen samples were collected from 0 infertile men and divided into three sections. The first section was used for semen analysis, the second section for polymerase chain reaction (PCR) in which U4 and U5 primers were used for the amplification of the urease gene of U. urealyticum, and RNAH and RNAH primers were used for amplification of the 6S rRNA gene of M. hominis. From a total of 0 semen samples cultured, 5.5% of M. hominis and 40.5% of U. urealyticum were isolated. Evaluation of semen parameters showed a lower pH in the U. urealyticum positive group and the group which was positive for both bacteria, rather than the group which contained no bacteria (p=0.007 and p=0.000, respectively). Also, the mean sperm motility was lower in the group which was positive for both bacteria when compared with the U. urealyticum positive group (p=0.009). The results of this study show that a high percent of infertile men are infected with these bacteria which may lead to pelvic inflammatory disease (PID) and infertility, thus isolation of these bacteria in infertile couples with no clinical symptoms is necessary and can be a part of a sexual transmitted disease (STD) control program.

The treatment efficiency of electrochemotherapy (ECT) or the use of a chemotherapy agent with a high electric field and low frequency has been reported. Unfortunately this protocol induces an unpleasant sensation to the patient. Therefore, the aim of this study was to investigate the efficiency of combined low electric field and high repetition frequency for the treatment of an animal tumor model, invasive ductal carcinoma.

Female Balb/c mice were transplanted with invasive ductal carcinoma. ECT with bleomycin and two different electric pulse simulation protocols were used. In the first protocol, eight high pulses at an amplitude of 000 V/cm with 00 µs duration and repetition frequencies of both 5 kHz and Hz were delivered. In the second protocol, low pulse amplitude of 00 V/cm with 5 kHz frequency and different numbers of pulses 500 (pulse with 50 milliseconds duration), 000 (4 pulses with 50 milliseconds duration), 4000 (8 pulses with 50 milliseconds duration) and 5000 (0 pulses with 50 millisecond duration) at 00 µs were applied.

ECT with a higher repetition frequency of electric pulses and low voltage inhibits tumor growth and has a comparable effect to the 5 kHz and Hz pulse repetitions at high voltage. Based on the results, the best antitumor effect was obtained at 4000 pulses or higher, with high frequency and low voltage (p<0.05). The rate of inhibition of tumor growth statistically increased with electric pulse numbers higher than 000.

The finding indicated that ECT with the use of low pulse amplitude and high frequency, combined with the best number of pulses has a comparable effect to a clinical protocol.

Human mesenchymal stem cells (MSCs) have been recognized as potential candidates for cell therapy. In the present study, the ability of human bone marrow mesenchymal stem cells (hBMSCs) to differentiate into cells with characteristics of cardiomyocytes in vitro was investigated.

hBMSCs cultured in enriched medium were treated with oxytocin and 5-azacytidin. The differentiation of hBMSCs into cells that expressed cardiac-specific genes such as α-actinin, alpha - myosin heavy chain (α-MHC), beta - myosin heavy chain (β-MHC), myosin light chain isoform a (MLCa), myosin light chain isoform v (MLCv), artial natriuretic factor (ANF), GATA4 and oxytocin receptor (OTR) was investigated by reverse transcription-polymerase chain reaction (RT-PCR). Protein expressions of β-actinin and troponin I-C in the cells were analyzed through immunofluorescence staining.

MSCs are spindle-shaped with irregular processes. Cells treated with oxytocin and 5-azacytidin connected with adjoining cells to form myotube-like structures. Expressions of a number of cardiac-specific genes were detected by RT-PCR. Immunofluorescence staining analysis showed that the differentiated cells stained positively for β-actinin and troponin I-C protein.

These results indicate that adult hBMSCs can differentiate into cardiomyocytes in vitro by treatment with oxytocin and 5-azacytidin, and can be considered as a source of cells for cellular cardiomyoplasty.

In this study we have investigated the changes in D receptor expression level in morphine-sensitized mice, in the absence and presence of lithium chloride (LiCl). The result would pave the way to comprehend and confront this complicated event.

Male NMRI mice, weighing 0-5g, were used in this study. They were divided into six groups. The first group received 0.9% saline as the control group and the other group was treated with morphine sulphate (0 mg/kg). LiCl (5 and 0 mg/kg treatments) was separately performed in two other groups. The final two groups were simultaneously treated with morphine sulphate (0 mg/kg) and LiCl (5 mg/kg) in one group and morphine sulphate (0 mg/kg) accompanied by LiCl (0 mg/kg) in the other group. All injections were performed intraperitoneally and once daily. After a five day wash-out, mice were decapitated and the brain regions which included the striatum, prefrontal cortex (PFC) and hippocampus were extracted. Using relative Real-Time polymerase chain reaction (PCR), the expression levels of the long (DL) and short (DS) isoforms of the D receptor were investigated.

Morphine treatment leads to a significant increase (p<0.0.5) in DS levels in the striatum and PFC but has no effect on DL levels in the examined regions. In the group receiving LiCl 5mg/kg, the DL levels showed a significant augmentation in PFC and the hippocampus (p<0.05) as well as the striatum (p<0.00). The DS levels in the same group, significantly increased in the PFC (p<0.05) and striatum (p<0.00). LiCl at a dose of 0 mg/kg did not alter the expression of either isoforms in any region. While simultaneous administration of morphine and LiCl (0 mg/kg) resulted in a marked increase in DS levels in the striatum (p<0.00) and PFC (p<0.05), morphine administration along with LiCl (5mg/kg) was ineffective on the expression levels of DL and DS isoforms when compared to the control group.

Morphine sensitization leads to an increase in DS receptor expression and is affected by lithium in a dose-dependent manner where the lower dose inhibits and higher dose to enhances this effect. It is assumed that sensitization-induced changes in the transcript level are more regulated by dopamine transmission rather than by the direct effect of morphine and/or lithium on gene expression.

Diazinon (DZN) and Hinosan, two of the most important organophosphate pesticides, are widely used for pest control in gardens and agriculture. These compounds adversely effect enzyme activation and reproductive organs. Therefore, in the present study, we investigated DZN and Hinosan impacts on the structure of testis tissue and sexual hormones in mice. For this study, 60 male Balb/C mice were divided into the following groups: DZN, Hinosan, control and sham. In the experimental groups, mice were injected with a single dose of DZN (0 mg/kg intraperitoneal) and Hinosan (0 mg/kg intraperitoneal), sham (corn oil) and control (no injection). Animals were sacrificed 5 days after the latest injection. Blood samples were collected and testosterone, luteinzing hormones (LH) and Follicle stimulating hormone (FSH) levels were assayed. Testis tissues sections were provided to investigate the changes of spermatogenic and Leydig cells. Testis and seminiferous diameter were assayed with micrometer and eye piece, respectively. Data were analyzed with one-way ANOVA. Significance was set at p<0.05. In the present study, no significant differences in testis and seminiferous diameter and number of blood vessels were noted. However the number of germ cells, spermatocysts, spermatids and Leydig cells on the testes of mice decreased (p<0.05). There was no significant difference between the parameters of the sham group compared to the control group. These results suggest that DZN and Hinosan can exert a decrease in sperm production. Therefore, application of DZN should be limited to a designed program.

Assessment of functional recovery of neural stem cells (NSCs) and (-)-deprenyl, in a contusive spinal cord injury model in rats. A total of 4 female Sprague Dawley rats were randomly, but equally (n=6), allocated into the following groups: control, sham, NSC graft and NSC graft + (-)-deprenyl. All animals were laminectomized at the T level. Contusion was performed according to the weight dropping technique in the control, NSC and NSC graft + (-)-deprenyl groups. Daily injections of 0. mg/kg (ip) (-)-deprenyl were administered to the NSC graft + (-)-deprenyl group and an equal amount of saline into the other groups for 4 days. The NSC graft and NSC graft + (-)-deprenyl groups received stereotaxic injections of 00,000 labelled NSCs at day nine after injury. Behavioral Basso, Beattie and Bresnahan (BBB) test was carried out in all groups at day one (after the contusion day) and at the end of each week for eight weeks. In addition, cavity and spared tissue volume at the site of injury and number of motoneurons at frozen sections of spinal cord were obtained and compared by ANOVA. Differentiation of grafted NSCs into astrocytes, oligodendrocytes and neurons were evaluated by immunohistochemistry. Motor ability of the NSC graft + (-)-deprenyl group in comparison with the control and NSCs groups increased significantly at the end of the study. The mean volume of spared spinal cord and mean number of motoneurons significantly increased in the NSCs and NSC graft + (-)-deprenyl groups compared with the control group. Immunohistochemical evaluations revealed that the grafted NSCs were alive at the end of the study and differentiated into astrocyes, oligodendrocytes and neurons in both the NSCs and NSC graft + (-)-deprenyl groups. In addition, in the NSCs graft group, transplanted cells mainly concentrated around the cavity and showed less differentiation, while in the NSCs graft + (-)-deprenyl group transplanted cells were more scattered and differentiated into one of the above mentioned cell lines. The results of the present study indicate that (-)-deprenyl and NSCs, probably via protection of motoneurons, spinal cord tissue and replacement of lost cells, improves motor recovery in a contusive SCI model in rats.

Duchenne and Becker Muscular Dystrophy (DMD and BMD) are X-linked conditions resulting from a defect in the dystrophin gene located at Xp.. DMD is the most frequent neuromuscular disease in humans (/500 male newborns). In approximately 65% of DMD and BMD patients, deletions in the dystrophin gene have been identified as the molecular determinant. The frequency and distribution of dystrophin gene deletions in DMD/BMD patients from different populations are different. The aim of this study was to delineate various types of deleted exons and their frequency in affected male patients and identification of carrier females by linkage analysis. In this study 00 unrelated patients with DMD/BMD were studied for intragenic deletions in 8 exons and the promoter region of the dystrophin gene using multiplex PCR. We also performed linkage analysis within the dystrophin gene utilizing 8 short tandem repeat markers. Fifty-two (5%) patients showed intragenic deletions. A total of 8% of the deletions were located at the distal hot spot region (44-55 exons) and 9% of the deletions were located at the proximal region (exon -9). The most frequent deleted exons were 47(6%), 48 and 46 (%). Most of the STR markers showed heterozygosity in the families studied. The linkage analysis was useful for detecting carrier status. The present study suggests that intragenic dystrophin gene deletions occur with the same frequency in Iranian patients compared with other ethnic groups.

The main objective of this study was to compare the quality and effectiveness of the sedimentation velocity at unit gravity (SVUG) method with the percoll density gradient centrifugation method in the separation of spermatogenic cells in order to introduce a simple and cost-effective method. In this study, the testes from male mice (NMRI strain) were obtained and transferred to Ham's F0 medium. With the use of the mechanical and enzymatic digestion method, spermatogenic cells were isolated. Spermatogenic cell separation was undertaken with both the percoll gradient centrifugation and sedimentation velocity at unit gravity (SVUG) methods based on human serum albumin (HSA) gradients. Subsequently the May-Grunwald-Giemsa (MGG) staining method was used to identify isolated cells. The combined method possibly could isolate a purer fraction of spermatogenic cells. This method (SVUG and Percoll Gradients Centrifugation combination) has the potential to be used as a simple and effective method for the isolation of spermatogenic cells for diagnostic or research purposes.