DNA Polymerase gamma ; DNA-Directed DNA Polymerase ; genetics ; Diffuse Cerebral Sclerosis of Schilder ; diagnosis ; drug therapy ; etiology ; genetics ; Heterozygote ; Humans ; Infant ; Male ; Mutation

The objective of this study was to determine the effect of ionizing radiation (IR) exposure of parents on carcinogenesis of the next generation focusing on the epigenetic perspective to clarify the relationship between radiation dose and carcinogenesis in F1 generation SD rats. F1 generations from pregnant rats (F0) who were exposed to gamma rays were divided into three groups according to the dose of radiation: 10 rad, 30 rad, and untreated. They were intraperitoneally injected with 50 mg/kg of diethylnitrosamine (DEN). Carcinogenesis was analyzed by examining expression levels of tumor suppressor genes (TSG) and other related genes by methylation-specific polymerase chain reaction (MSP). DNA methylation in liver tissues was evaluated to discern epigenetic regulation of transgenerational carcinogenesis vulnerability following IR exposure. Numerous studies have proved that transcriptional inactivation due to hypermethylation of TSG preceded carcinogenesis. Results of this study revealed hypermethylation of tumor suppressor gene SOCS1 in group treated with 30 rad. In addition, genes related to DNA damage response pathway (GSTP1, ATM, DGKA, PARP1, and SIRT6) were epigenetically inactivated in all DEN treated groups. In the case of proto-oncogene c-Myc, DNA hypermethylation was identified in the group with low dose of IR (10 rad). Results of this study indicated that each TSG had different radiation threshold level (dose-independent way) and DEN treatment could affect DNA methylation profile irrelevant of ionizing radiation dose.
Animals ; Carcinogenesis* ; Diethylnitrosamine ; DNA ; DNA Damage ; DNA Methylation ; Epigenomics* ; Family Characteristics ; Gamma Rays ; Genes, Tumor Suppressor ; Humans ; Liver ; Parents ; Polymerase Chain Reaction ; Proto-Oncogenes ; Radiation, Ionizing* ; Rats

To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.
Bone Marrow ; Cell Culture Techniques* ; Cell Lineage ; Gene Expression ; Humans ; PPAR gamma ; RNA-Directed DNA Polymerase ; Tissue Donors ; Tissue Engineering*

OBJECTIVETo analyze the polymerase gamma(Polg) gene polymorphisms in Chinese idiopathic infertile males and evaluate the correlation of the polymorphisms with male infertility.

METHODSWe conducted a study of Polg CAG repeats in the sperm or blood DNA of 55 asthenospermia patients, 57 oligospermia patients, 34 azoospermia patients, and 104 controls with PCR and GeneScan. Phenotypic data were available in all the subjects, including semen parameters, and clinical profiles.

RESULTSThe frequency of 10/not 10 CAG genotype in asthenospermia patients was higher than in the other groups, but with no significance.

CONCLUSIONOur findings have shown for the first time that there exits an ethnic difference between Chinese and European males in the number of CAG repeats of Polg gene, and that 10/not 10 CAG genotype may affect sperm motility.

Adult ; Case-Control Studies ; China ; epidemiology ; DNA Polymerase gamma ; DNA-Directed DNA Polymerase ; genetics ; Gene Frequency ; Genotype ; Humans ; Infertility, Male ; epidemiology ; ethnology ; genetics ; Male ; Polymerase Chain Reaction ; Trinucleotide Repeats

PURPOSE: Epidemiologic evidence suggests an infectious cause for Kawasaki disease(KD), but the etiology of KD remains unknown. The aim of this study was to determine whether viral infections are related in the pathogenesis of KD by detecting viral genomes using nested polymerase chain reaction. METHODS: Peripheral blood was obtained from 18 acute KD patients before administration of intravenous gamma globuline and 11 age-matched control patients. DNA extraction from whole blood was performed using proteinase K and phenol. The primer sequences were derived frorn UL-42 for herpes simplex virus type 1(HSV-1), US-4 for herpes simplex virus type 2(HSV-2), major IE for cytornegalovirus(CMV) and IR1 for Epstein-Barr virus(EBV). Two successive PCR were performed, and the PCR products were detected by agarose gel electrophoresis. RESULTS: HSV-1-PCR was negative in both groups and HSV-2-PCR was positive in 2 of 17 of the KD group. CMV-PCR was positive in 1 of 11 in the control group and all negative in the KD group. EBV-PCR was positive in 7 of 18(39%) of the KD group and in 2 of 11(18%) of the control group. EBVCA-IgM was negative in all 5 EBV-PCR positive patients, but EBVCA-IgG, EBNA and EBEA was positive in 1 EBV-PCR positive patient. CONCLUSION: This study suggests that an unusual EBV-cell interaction may exist in Kawasaki disease.
DNA ; DNA, Viral* ; Electrophoresis, Agar Gel ; Endopeptidase K ; gamma-Globulins ; Genome, Viral ; Herpesvirus 4, Human ; Humans ; Mucocutaneous Lymph Node Syndrome* ; Phenol ; Polymerase Chain Reaction* ; Simplexvirus

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M₂ plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M₂ rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.
Crops, Agricultural/radiation effects* ; Gamma Rays ; Genetic Techniques ; Genome, Plant ; Homozygote ; INDEL Mutation ; Mutagenesis ; Oryza/radiation effects* ; Plant Breeding ; Polymerase Chain Reaction ; Seeds ; Sequence Analysis, DNA ; Sequence Deletion

OBJECTIVETo evaluate the practical values of PCR detectable T-cell receptor (TCR) gene rearrangement in paraffin embedded tissue samples in the diagnosis of T-cell malignancies using BIOMED-2 PCR multiplex tubes TCRgamma(A+B).

METHODSTraditional phenol-chloroform method was used to extract DNA from 55 cases of archival paraffin embedded tissues samples of T-cell malignancies and the DNA quality was evaluated by PCR-based amplification of housekeeping gene beta-globin. The selected BIOMED-2 PCR multiplex tubes TCRgamma(A+B) were used to detect TCR gene rearrangement and comparison with the results of universal TCR primers (T(VG)/T(JX)) was performed.

RESULTSPositive detection rates by the BIOMED-2 multiplex tubes TCRgamma(A+B) and the universal primers (T(VG)/T(JX)) were 76.4% and 60.0%, respectively. There were not statistical difference between the methods (P > 0.05).

CONCLUSIONBIOMED-2 multiplex tubes TCRgamma(A+B) is suitable for detection of clonal rearrangements of TCR genes in current archival paraffin embedded tissue samples of T-cell malignancies.

DNA Primers ; DNA, Neoplasm ; analysis ; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor ; Humans ; Lymphoma, T-Cell ; genetics ; metabolism ; pathology ; Paraffin Embedding ; Polymerase Chain Reaction ; methods ; beta-Globins ; metabolism

The studies have provided the first comprehensive comparison of the factors regulating activation and proliferation of WC1+ and WC1- gammadelta T cells. The investigation has shown that accessory molecules essential for activation and function of WC1+ and WC1- gammadelta T cells and the sources and roles of cytokines in activation of gammadelta T cells through the T cell receptor (TCR). The study has also shown that the role of cytokines in activation and function of gammadelta T cells activated indirectly through cytokines secreted by ab T cells, accessory cells and antigen presenting cells (APC). Cytokines were differentially produced by subpopulations of gammadelta T cells under different conditions of activation. The investigation obtained in this study has revealed that factors account for activation and proliferation of gammadelta T cells in cultures designed to study MHC-restricted responses to antigens. Evidence obtained here has shown there is biological relevance to activation under these culture conditions that points to potential regulatory and effector functions of gammadelta T cells. The investigations have also provided the information needed to begin identifying and characterizing antigens recognized by the TCR repertoires of WC1+ and WC1- gammadelta T cells. Finally, the investigations have provided the information needed to begin analysis of the mechanisms by which gammadelta T cells modulate MHC restricted immune responses to pathogens and derived vaccines.
Animals ; Base Sequence ; Cattle ; Concanavalin A ; Cytokines/genetics/immunology ; DNA Primers ; Immunophenotyping ; Lymph Nodes/immunology ; Lymphocyte Activation ; Receptors, Antigen, T-Cell, gamma-delta/*immunology ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes/classification/*immunology

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is common disorder of the school-age population. ADHD is familial and genetic studies estimate heritability at 80–90%. The aim of the present study was to investigate the association between the genetic type and alleles for gamma-aminobutyric acid receptor subunit beta-3 (GABA3) gene in Korean children with ADHD. METHODS: The sample consisted of 180 ADHD children and 159 control children. We diagnosed ADHD according to DSM-IV. ADHD symptoms were evaluated with Conners' Parent Rating Scales and Dupaul Parent ADHD Rating Scales. Blood samples were taken from the 339 subjects, DNA was extracted from blood lymphocytes, and PCR was performed for GABA3 rs2081648, rs1426217 and rs981778 Polymorphism. Alleles and genotype frequencies were compared using the chi-square test. We compared the allele and genotype frequencies of GABA3 gene polymorphism in the ADHD and control groups. RESULTS: This study showed that there was a significant correlation among the frequencies of the rs2081648 (OR=0.71, 95% CI=0.51–0.98, p=0.040) of alleles of MAO, but the final conclusions are not definite. Follow up studies with larger patient or pure subgroups are expected. CONCLUSION: These results suggested that GABA3 might be related to ADHD symptoms.
Alleles ; Attention Deficit Disorder with Hyperactivity* ; Child* ; Diagnostic and Statistical Manual of Mental Disorders ; DNA ; Follow-Up Studies ; gamma-Aminobutyric Acid ; Genotype ; Humans ; Lymphocytes ; Monoamine Oxidase ; Parents ; Polymerase Chain Reaction ; Receptors, GABA ; Weights and Measures

OBJECTIVE: Tributyltin (TBT), an endocrine disrupting chemical, has been reported to decrease ovarian function by causing apoptosis in the ovary, but the mechanism is not fully understood. Therefore, we examined whether TBT increases the expression of adipogenesis-related genes in the ovary and the increased expression of these genes is associated with apoptosis induction. METHODS: Three-week-old Sprague-Dawley rats were orally administered TBT (1 or 10 mg/kg body weight) or sesame oil as a control for 7 days. The ovaries were obtained and weighed on day 8, and then they were fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or frozen for RNA extraction. Using the total RNA of the ovaries, adipogenesis- and apoptosis-related genes were analyzed by real-time polymerase chain reaction (PCR). RESULTS: The ovarian weight was significantly decreased in rats administered 10 mg/kg TBT compared to that in control rats. As determined by the TUNEL assay, the number of apoptotic follicles in ovary was significantly increased in rats administered 10 mg/kg TBT. The real-time PCR results showed that the expression of adipogenesis-related genes such as PPARgamma, aP2, CD36, and PEPCK was increased after TBT administration. In addition, apoptosis-related genes such as TNFalpha and TNFR1 were expressed more in the TBT-administered rats compared with the control rats. CONCLUSION: The present study demonstrates that TBT induces the expression of adipogenesis- and apoptosis-related genes in the ovary leading to apoptosis in the ovarian follicles. These results suggest that the increased expression of adipogenesis-related genes in the ovary by TBT exposure might induce apoptosis resulting in a loss of ovarian function.
Adipogenesis ; Animals ; Apoptosis ; DNA Nucleotidylexotransferase ; Female ; In Situ Nick-End Labeling ; Ovarian Follicle ; Ovary ; PPAR gamma ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor, Type I ; RNA ; Sesame Oil ; Trialkyltin Compounds ; Tumor Necrosis Factor-alpha