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

OBJECTIVETo explore the expression of phospholipase C-gamma 1 (PLC-gamma1) alternative splicing variants in rats.

METHODSAccording to the sequence of human PLCG1 splicing variant, specific primers for rat PLC-gamma1 were designed and synthesized. The rat RNA was reverse transcribed into cDNA, which was amplified using the specific primers, and the PCR products were sequenced and analyzed using BLAST and bioinformatics methods. Totally 21 rat tissue samples were examined, including the heart, liver, lung, kidney, eyeball, and brain obtained in 3 different embryonic stages, 7 different early postnatal stages, and in adulthood.

RESULTSThe result did not show that rat PLC-gamma1 had the same splicing variant (PLC-gamma1a, NM_002660) as human does.

CONCLUSIONSThe same splicing variant of PLC-gamma1 detectable in human may not exist in rats, and the pre-mRNA may undergo splicing resulting predominantly in PLC-gamma1b mRNA. Very likely, the alternative splicing site of rat PLC-gamma1 is not identical to that of human.

Alternative Splicing ; Animals ; Base Sequence ; Molecular Sequence Data ; Phospholipase C gamma ; genetics ; RNA Precursors ; genetics ; RNA Splice Sites ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo establish an accurate and efficient method for detecting recent thymic output function and analyze the content of T-cell receptor (TCR) rearrangement excision circles (TRECs) within peripheral blood mononuclear cells (PBMCs).

METHODSAccording to the specific sequence of TCRdelta, the primers and the fluorescent probe (TaqMan) were designed and synthesized. The standard quantitative template was constructed by T/A cloning. The method for detecting TRECs was established after optimization of reaction condition, then its specificity, sensitivity and stability were tested. Quantitative detection of TRECs in DNA of PBMCs from normal individuals and patients of chronic hepatitis B were preformed by real-time PCR using TaqMan technique.

RESULTSDetection of TRECs was quick and accurate by real-time fluorescence quantitative PCR. The CV value of Ct was 1.06%, the product was specific which was confirmed by electrophoresis and sequencing and the method showed high sensitivity. The mean value of TRECs from normal individuals was (7767.4 +/- 2369.5) copies/10(6)PBMCs in healthy controls at age 21.45 but (28,374.4 +/- 7820.4) copies/10(6)PBMCs in those at age 16.20 (P < 0.05). The mean value of TRECs from patients with chronic hepatitis B was (6480.9 +/- 2031.2) copies/10(6) PBMCs in those at age 21.45, which was statistically significant as compared with normal individuals at age 21.45.

CONCLUSIONReal-time fluorescence quantitative PCR for detecting the TRECs is an accurate, efficient and stable method and the recent thymic output function might decrease in patients with chronic hepatitis B.

Adult ; DNA Primers ; Female ; Gene Rearrangement, T-Lymphocyte ; Hepatitis B, Chronic ; blood ; genetics ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Receptors, Antigen, T-Cell, gamma-delta ; genetics ; Reproducibility of Results ; Thymus Gland ; immunology ; metabolism ; Young Adult

PURPOSE: The channelopathies has been implicated in the pathogenesis of idiopathic generalized epilepsy(IGE). Recently, nonsense and missense mutations in a voltage-gated chloride channel gene(CLCN2) have been shown to be associated with IGE. Loss of CLCN2 function results in loss of sustained GABA inhibition, increasing the risk of uncontrolled firing leading to seizure activity. This study assessed the relevance of mutations in the CLCN2 gene in the Korean patients with IGE. METHODS: Twenty seven patients with IGE were recruited in the Seoul National University Boramae Hospital. PCR and direct sequencing of genomic DNA were done to analyze the complete coding region of CLCN2. 99 controls were tested for two identified polymorphisms. Genotypes and allelic frequencies were compared to controls with epilepsy patients and subgroup of IGE: 10 generalized epilepsy with febrile seizure plus(GEFSP), 9 childhood absence epilepsy(CAE) and 8 other IGE patients. RESULTS: Twelve CLCN2 polymorphisms: 3 exonic, 2 promotor and 7 intronic, were found in 22 patients(81%) and 2 polymorphisms were noble. Two polymorphisms in the exonic region with changes of amino acid, p.L15P and p.T668S and 2 polymorphisms in the promoter regions(c.1-1990T>C, c.1-693G>A) may affect on the CLCN channel function. The odds ratio for developing other IGE in patients with RS9820367-CG type was 4.2 compared to individuals with CC type. In addition, the odds ratio for developing other GEFSP in patients with RS9820367-CC type was 4.0 compared with individuals with CG type. CONCLUSION: Our findings suggest that genomic variations of CLCN2 may be implicated in the pathogenesis of IGE.
Channelopathies ; Chloride Channels ; Clinical Coding ; DNA ; Epilepsy ; Epilepsy, Generalized ; Exons ; Fires ; gamma-Aminobutyric Acid ; Genotype ; Humans ; Immunoglobulin E ; Introns ; Mutation, Missense ; Odds Ratio ; Polymerase Chain Reaction ; Seizures ; Seizures, Febrile