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Polymerase Chain Reaction Method (polymerase + chain_reaction_method)
Selected AbstractsAnalysis of growth hormone receptor polymorphism in Japanese semisuper centenariansGERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 2 2006Yuchen Du Background: Recent studies have demonstrated a significant association between mutations in genes involved in the GHR/IGF1 signaling pathway and extension of the lifespan of model organisms. Exon 3 insertion or deletion is one common polymorphism in the growth hormone receptor (GHR) of humans. The exon 3 deletion allele is reported to have stronger signaling in the GH/GHR pathway, which may correlate to short lifespan. Methods: We investigated the common polymorphic variation in 119 Japanese semisuper centenarians (SSC; older than 105) compared with 104 healthy younger controls via the polymorphism-based polymerase chain reaction method. Results: The frequency of exon 3 deletion variation of GHR in SSC was found to be higher than controls, although this was not significant statistically. Also, the single nucleotide polymorphism genotype frequency and allele frequency exhibited no differences between SSC and controls. Conclusions: These results show that SSC in Japan do not tend to have the allele of GHR, which has a lower signaling capacity. [source] Shorter Telomere Length in Peripheral Blood Cells Associated With Migraine in WomenHEADACHE, Issue 6 2010Hua Ren PhD (Headache 2010;50:965-972) Objective., To evaluate relative telomere length of female migraine patients. Background., Migraine is a debilitating disorder affecting 6-28% of the population. Studies on the mechanisms of migraine have demonstrated genetic causes but the pathophysiology and subcellular effects of the disease remain poorly understood. Shortened telomere length is associated with age-related or chronic diseases, and induced stresses. Migraine attacks may impart significant stress on cellular function, thus this study investigates a correlation between shortening of telomeres and migraine. Methods., Relative telomere length was measured using a previously described quantitative polymerase chain reaction method. A regression analysis was performed to assess differences in mean relative telomere length between migraine patients and healthy controls. Results., The leukocyte telomeres of a cohort of 142 Caucasian female migraine subjects aged 18-77 years and 143 matched 17-77-year-old healthy control Caucasian women were examined. A significantly shorter relative telomere length was observed in the migraine group compared with the control group after adjusting for age and body mass index (P = .001). In addition, age of onset was observed to associate with the loss of relative telomere length, especially at early age of onset (<17 years old). No association was observed between relative telomere length and the severity and frequency of migraine attacks and the duration of migraine. Conclusion., Telomeres are shorter in migraine patients and there is more variation in telomere length in migraine patients. [source] A NOVEL MULTIPLEX POLYMERASE CHAIN REACTION FOR SIMULTANEOUS DETECTION OF YERSINIA ENTEROCOLITICA, STAPHYLOCOCCUS AUREUS, AEROMONAS AND SALMONELLA FROM CHICKEN MEAT AND MILK SAMPLESJOURNAL OF FOOD SAFETY, Issue 2 2010K. BALAKRISHNA ABSTRACT Yersinia enterocolitica, Staphylococcus aureus, Aeromonas and Salmonella are among the most important foodborne bacterial pathogens. The majority of human infections caused by all of these organisms are associated with ingestion of undercooked and contaminated meat, dairy products and water where in the secreted bacterial toxins lead to foodborne intoxications. We, here, report a new multiplex polymerase chain reaction (mPCR) assay for the simultaneous detection of these important foodborne bacterial pathogens. The mPCR targeted Ail and virF genes of Y. enterocolitica, nuc and entB genes of S. aureus, aerA and 16S rRNA genes of Aeromonas and invA, an invasion protein A gene of Salmonella. An internal amplification control designed to check the false negative reactions in mPCR was also included. This procedure could detect initial populations of 1,100 cfu/g or /mL within 24 h in experimentally spiked food and water samples. When evaluated on a total of 104 naturally occurring food samples, the mPCR detected two samples to contain S. aureus, one was identified to contain Y. enterocolitica and four samples were identified to contain Salmonella species individually. This was compared with the standard microbiological and biochemical identification procedures. PRACTICAL APPLICATIONS All the microorganisms selected in this study are food and waterborne and contaminate a variety of food items. Pathogenic Y. enterocolitica and Aeromonas species are able to grow and multiply and secrete toxins even at low temperatures. The high throughput and cost-effective multiplex polymerase chain reaction method reported here could be a viable alternative for detection of pathogenic Y. enterocolitica, S. aureus, Aeromonas and Salmonella from food and environmental samples. [source] Effect of Chronic Ethanol Ingestion and Gender on Heart Left Ventricular p53 Gene ExpressionALCOHOLISM, Issue 8 2005Heidi Jänkälä Background: Although the beneficial effects of mild to moderate ethanol consumption have been implied with respect to heart, alcohol abuse has proven to be a major cause of nonischemic cardiomyopathy in Western society. However, the biochemical and molecular mechanisms, which mediate the pathologic cardiac effects of ethanol, remain largely unknown. The aim of the present study was to explore the effects of chronic ethanol exposure on cardiac apoptosis and expression of some of the genes associated with cardiac remodeling in vivo. Methods: Alcohol-avoiding Alko Non Alcohol rats of both sexes were used. The ethanol-exposed rats (females, n= 6; males, n= 8) were given 12% (v/v) ethanol as the only available fluid from age of three to 24 months of age. The control rats (females, n= 7; males, n= 5) had only water available. At the end of the experiment, free walls of left ventricles of hearts were immediately frozen. Cytosolic DNA fragmentation, reflecting apoptosis, was measured using a commercial quantitative sandwich enzyme-linked immunosorbent assay kit, and mRNA levels were analyzed using a quantitative reverse transcriptase,polymerase chain reaction method. Results: Ethanol treatment for two years increased cardiac left ventricular p53 mRNA levels significantly (p= 0.014) compared with control rats. The gene expression was also dependent on the gender (p= 0.001), so that male rats had higher left ventricular p53 mRNA levels than female rats. However, no significant differences in levels of DNA fragmentation were detected. Conclusions: Chronic ethanol exposure in vivo induces rat cardiac left ventricular p53 gene expression. Expression of p53 is also gender-dependent, males having higher p53 mRNA levels than females. This preliminary finding suggests a role for the p53 gene in ethanol-induced cardiac remodeling. The results might also have some relevance for the known gender-dependent differences in propensity to cardiovascular disease. [source] Isolation and characterization of microsatellite loci from Larix kaempferiMOLECULAR ECOLOGY RESOURCES, Issue 3 2006KEIYA ISODA Abstract Microsatellites were isolated and characterized for Japanese larch, Larix kaempferi, a conifer species distributed in Japan. A larch genomic DNA library enriched for (AG)n repeats was screened using the colony polymerase chain reaction method and 145 unique microsatellite containing sequences were obtained. Seventy-two primer pairs were designed and 30 produced single-locus products, and 19 of them were polymorphic. The expected heterozygosity ranged from 0.566 to 0.951. These 19 polymorphic microsatellite loci should be valuable markers for genetic studies on Japanese larch. [source] Relationship of Porphyromonas gingivalis with glycemic level in patients with type 2 diabetes following periodontal treatmentMOLECULAR ORAL MICROBIOLOGY, Issue 4 2008N. Makiura Introduction:, The aim of this study was to assess the relationship between serum glycemic levels and subgingival microbial profile alteration following periodontal treatment in patients with type 2 diabetes mellitus. Methods:, We studied 30 periodontitis patients with type 2 diabetes mellitus who received full-mouth subgingival debridement by analyzing their subgingival microbial profiles using a polymerase chain reaction method at baseline and various time-points for 12 months following treatment. Concurrently, probing pocket depth, bleeding on probing, and metabolic parameters, including glycated hemoglobin A1c (HbA1c), blood sugar level, C-reactive proteins, total cholesterol, triglyceride, and high-density and low-density lipoprotein cholesterol, were recorded. Results:, Periodontal conditions were significantly improved after treatment, and the occurrence rates of periodontal bacterial species, including Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, and Prevotella intermedia, were also reduced. Interestingly, P. gingivalis was detected more frequently in subjects with increased HbA1c values after periodontal treatment than in those patients with decreased HbA1c values. Furthermore, P. gingivalis with type II fimbriae was detected only in HbA1c-increased subjects, while improvements in HbA1c values were observed only in subjects without type II clones. Conclusions:, These results suggest that glycemic level in diabetes is affected by the persistence of P. gingivalis, especially clones with type II fimbriae, in periodontal pockets. [source] A new standard for the assessment of disease progression in murine cutaneous leishmaniasisPARASITE IMMUNOLOGY, Issue 5 2000Lynden J.roberts Infection of mice with Leishmania major has been used both as a model for the cutaneous disease in humans and as a model for the more general control and function of helper T cells in immunity. In both cases, disease patterns and disease progression have been assessed by two complementary methods, lesion size and parasite burden in the draining lymph nodes. We propose a much improved method for the graphical representation of lesion development which conveys more information with better accuracy. We also describe a polymerase chain reaction method for determining parasite burden, which is faster and allows the analysis of larger numbers of experimental animals than the current limiting dilution analysis. Moreover, these methods are equally applicable to other infectious diseases, an obvious one being schistosomiasis. [source] Solitary squamous cell papilloma of the lung in a 40-year-old woman with recurrent laryngeal papillomatosisPATHOLOGY INTERNATIONAL, Issue 5 2000Hidekazu Harada A rare case of recurrent respiratory papillomatosis (RRP) is reported with a review of the literature. A 40-year-old Japanese woman had suffered from RRP since 1 year of age. She developed a pulmonary squamous papilloma with a thin-walled cavity, which was suspected as being lung carcinoma. The trachea and bronchi around the tumor were intact, and no malignant transformation was present. Two types of human papillomavirus, 6 and 16, were detected, both in the laryngeal and pulmonary papillomas by in situ hybridization and the polymerase chain reaction method. To date, only 40 cases of juvenile laryngeal papilloma with pulmonary involvement have been reported in the English literature. [source] Response of anti-oxidant enzymes mRNA in the neonatal rat liver exposed to 1,2,3,4-tetrachlorodibenzo- p -dioxin via lactationPEDIATRICS INTERNATIONAL, Issue 5 2002Yumi Kono Abstract Background: The aim of this study was to assess the response to dioxin-induced oxidative stress in neonates via lactation in the model we have described previously. Methods: Maternal rats were treated with a single dose of 50 or 100 µmol/kg 1,2,3,4-tetrachlorodibenzo- p -dioxin (TCDD) on the first day postpartum (day 1). Messenger RNA levels of the key anti-oxidant enzymes (AOE), phospholipid hydroperoxide-glutathione peroxidase (PH-GPx), cellular-glutathione peroxidase (cell-GPx), copper-zinc superoxide dismutase (CuZn SOD), manganese superoxide dismutase (Mn SOD) and catalase (CAT) in the neonatal and maternal livers were determined by a competitive reverse transcription, polymerase chain reaction method. Results: Lactational transfer of 1,2,3,4-TCDD induced an inhibition of PH-GPx and cell-GPx mRNA in the neonatal liver on day 2 to 68 (P < 0.01) and 62% (P < 0.05) of the control at 100 µmol/kg, respectively. Both GPx mRNA returned to control levels on day 6 and thereafter increased to levels higher than the controls on day 10. In the dam rat, 10 days after the treatment, no remarkable change of PH-GPx or cell-GPx mRNA was observed. Copper-zinc superoxide dismutase and CAT mRNA of neonates on day 2 were also suppressed at 100 µmol/kg and then slightly increased on day 10. However, Mn SOD mRNA was not suppressed, but increased to a 2.1-fold level of the control (P < 0.05) on day 10 with 100 µmol/kg 1,2,3,4-TCDD. Conclusion: Quantitative analysis of AOE mRNA showed that PH-GPx and cell-GPx mRNA, as well as CuZn SOD and CAT mRNA in the neonatal liver were suppressed for a short period of time by 1,2,3,4-TCDD exposure via lactation. Dioxin induced oxidative stress by lactational transfer may alter pretranslation regulation of protective AOE in neonates. [source] Prefrontal cognition in schizophrenia and bipolar illness in relation to Val66Met polymorphism of the brain-derived neurotrophic factor genePSYCHIATRY AND CLINICAL NEUROSCIENCES, Issue 1 2006JANUSZ K. RYBAKOWSKI md Abstract, The measures of prefrontal cognition have been used as endophenotype in molecular-genetic studies. Brain-derived neurotrophic factor (BDNF) has been implicated in cognitive functions and in the pathogenesis of major psychoses. This study investigates the relationship between Val66Met polymorphisms of the BDNF gene and prefrontal cognitive function in 129 patients with schizophrenia and 111 patients with bipolar mood disorder. Cognitive tests included the Wisconsin Card Sorting Test (WCST), with such domains as number of perseverative errors, non-perseverative errors, completed corrected categories, conceptual level responses, and set to the first category, and the N-back test, where mean reaction time and percent of correct reactions were measured. Genotyping for Val66Met BDNF polymorphism was done by polymerase chain reaction method. In schizophrenia, no relationship between Val66Met polymorphism of the BDNF gene and the results of the WCST was observed. Patients with Val/Val genotype had a higher percentage of correct reactions in the N-back test than those with the remaining genotypes. Bipolar patients with Val/Val genotype obtained significantly better results on three of five domains of the WCST. No relationship between BDNF polymorphism and the results of the N-back test was found in this group. A limitation to the results could be variable psychopathological state and medication during cognitive testing and lack of Hardy,Weinberg equilibrium in schizophrenia group. Val66Met polymorphism of the BDNF gene may be associated with cognitive performance on the WCST in bipolar mood disorder but not in schizophrenia. An association of this polymorphism with performance on the N-back test in schizophrenia and not in bipolar illness may suggest that in schizophrenia, the BDNF system may be connected with early phases of information processing. [source] Contribution of a haplotype in the HLA region to anti,cyclic citrullinated peptide antibody positivity in rheumatoid arthritis, independently of HLA,DRB1ARTHRITIS & RHEUMATISM, Issue 12 2009Yukinori Okada Objective To examine the risk of anti,cyclic citrullinated peptide (anti-CCP) antibody positivity in rheumatoid arthritis (RA) patients carrying certain haplotypes in the HLA region. Methods A total of 1,389 Japanese patients with RA were genotyped for 30 single-nucleotide polymorphisms (SNPs) in the HLA region using commercial oligonucleotide arrays (from Perlegen or Affymetrix) as well as for HLA,DRB1 alleles using a sequence-specific polymerase chain reaction method. Stepwise logistic regression was used to select from among the 30 SNPs the ones that represented a risk of anti-CCP antibody positivity. Haplotypes of the selected SNPs were inferred using an expectation-maximization algorithm. Associations of individual SNPs were evaluated with the Cochran-Armitage test for trend. DRB1 alleles and haplotypes were evaluated with the chi-square test. Heterogeneities of risks among the shared epitope (SE) and non-SE HLA,DRB1 alleles were examined using the exact test. Haplotype associations that were independent of individual HLA,DRB1 alleles were evaluated using the likelihood ratio test. Results Significant associations were found for 9 SNPs (smallest P value being 2.4 × 10,8) and in 4 HLA,DRB1 alleles (smallest P value being 2.0 × 10,10 in DRB1*0405). Stepwise logistic regression selected 4 SNPs (rs9262638, rs7775228, rs4713580, and rs9277359). Among the 16 inferred haplotypes of these 4 SNPs, 6 indicated significant associations (smallest P value being 1.9 × 10,11). Risks among SE and non-SE alleles were significantly heterogeneous (P = 0.0095 and P = 9.8 × 10,9, respectively), indicating the importance of stratification with individual DRB1 alleles rather than SE alleles. Conditional analysis of the risk associated with individual DRB1 alleles identified a risk haplotype that was independent of DRB1 (odds ratio 2.00 [95% confidence interval 1.44,2.79], P = 2.6 × 10,5). Conclusion Heterogeneous risks of anti-CCP antibody positivity were confirmed among SE and non-SE alleles in our patient population. A risk haplotype in the HLA region that is independent of HLA,DRB1 was confirmed. [source] Expression and modulation of ghrelin O -acyltransferase in cultured chondrocytesARTHRITIS & RHEUMATISM, Issue 6 2009Rodolfo Gómez Objective To use reverse transcription,polymerase chain reaction to detect ghrelin O -acyltransferase (GOAT) transcripts in both murine and human chondrocytes, to evaluate the effect of pharmacologic in vitro treatments with lipopolysaccharide (LPS), growth hormone, ghrelin, and dexamethasone on GOAT messenger RNA (mRNA) expression, and to study the GOAT mRNA profile during chondrocyte differentiation. Methods Murine and human GOAT and ghrelin mRNA levels were determined by the SYBR Green,based quantitative real-time polymerase chain reaction method. Results GOAT mRNA was expressed in murine cartilage explants as well as in the cultured murine chondrogenic ATDC-5 cell line. GOAT was also expressed in human immortalized chondrocyte cell lines and in human cultured primary chondrocytes. In addition, GOAT mRNA expression in differentiating ATDC-5 cells was lower at the early stage of differentiation (days 3,7), whereas GOAT mRNA levels increased progressively at the late stages. Finally, among the drugs and hormones tested, only LPS was able to strongly decrease GOAT mRNA expression. Conclusion These data indicate that chondrocytes are equipped with biochemical machinery for the synthesis of acylated ghrelin and suggest a novel role of the ghrelin axis in prehypertrophic and hypertrophic chondrocyte differentiation during endochondral ossification. [source] Expression of Multidrug Resistance-related Transporters in Human Breast CarcinomaCANCER SCIENCE, Issue 4 2001Atsuko Kanzaki The expression levels of mRNA for multidrug resistance 1 (MDR1) gene, multidrug resistance protein 1 (MRP1), lung resistance-related protein (LRP) and breast cancer resistance protein (BCRP), which confer multidrug resistance in vitro, were examined in 43 untreated breast carcinoma patients, of whom 38 subsequently received doxorubicin-based chemotherapy after surgery, in order to elucidate the roles of these genes in drug resistance in vivo. The mRNA levels were determined using a semi-quantitative reverse-transcription polymerase chain reaction method in breast carcinoma tissues including at least 80% carcinoma cells. The expression level of BCRP gene was low and did not vary markedly in comparison with that of MDR1, MRP1 or LRP gene. The expressions of MDR1 and MRP1 genes were correlated with each other, but the expression of BCRP or LRP gene did not correlate with that of other genes. These four gene expressions were independent of age, TNM categories and the status of progesterone or estrogen receptor. The expression levels of these four genes were not related to the relapse or prognosis of the 38 patients treated with doxorubicin-based chemotherapy. P-glycoprotein (P-gp)/MDRl, MRP1 and LRP may play more important roles than BCRP in chemotherapy of human breast carcinoma. [source] The influence of antisense oligonucleotides against STAT5 on the regulation of normal haematopoiesis in a bone marrow modelCELL PROLIFERATION, Issue 3 2004M. Ba, kiewicz-Masiuk The STAT5 (signal transducers and activators of transcription) proteins are members of a family of signal transducers and activators of transcription that can be activated after cytokine stimulation. Their binding to promoters of different genes influences cell proliferation, differentiation and survival. It is suggested that they play an important role in haematopoiesis, however, the question of the real function of STAT5 proteins requires further examination. The aim of our study was to investigate the role of STAT5 in the proliferation and apoptosis of normal haematopoietic bone marrow cells derived from heparinized cadaveric organ donors (HCOD). We applied antisense oligodeoxynucleotides (ODNs) to block STAT5A and STAT5B at the mRNA level and the reverse transcription polymerase chain reaction method to study STAT5 mRNA expression in the cells after incubation with ODNs. Moreover, we performed Western blot analysis of the STAT5A protein after exposure to antisense STAT5A. We analysed the clonogenicity of the colony-forming unit of granulocytes,macrophages and the burst-forming unit of erythrocytes in methylcellulose cultures according to the type and the dose of ODNs. We also examined apoptosis induced in bone marrow mononuclear and CD34+ cells by employing annexin V staining and the TUNEL method using flow cytometry (FACScan). We found that the perturbation of STAT5 expression decreased the clonogenicity of bone marrow haematopoietic cells. However, we did not observe any significant increase in the percentage of apoptotic cells after incubation with antisense ODNs. It was concluded that the STAT5 proteins play a significant role in the proliferation of human bone marrow cells harvested from HCOD. These proteins might be critical in the regulation of haematopoiesis, especially under stress conditions. [source] Lack of correlation between fatty acid oxidation disorders and haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome?ACTA PAEDIATRICA, Issue 1 2005M. Holub Abstract Aim: Fatty acid ,-oxidation defects comprise a heterogeneous group of disorders that may precipitate acute life threatening metabolic crises particularly during catabolic episodes. Several studies have demonstrated a possible association between fatty acid ,-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and severe pregnancy complications. However, the precise percentage of women with haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome associated with foetal fatty acid ,-oxidation defects is not known. Methods: We carried out a multicentre retrospective study on 88 infants, born to women with HELLP syndrome. Acylcarnitine profiles from blood dried on filter paper cards were analysed by tandem mass spectrometry for the diagnosis of fatty acid ,-oxidation defects. In addition, we screened for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation using a standard restriction fragment length polymorphism polymerase chain reaction method. Results: None of the infants studied carried the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation. There was no evidence of fatty acid ,-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, as expected by unremarkable acylcarnitine profiles, while three infants with fatty acid ,-oxidation defects were diagnosed in the control group. Conclusions: Neither foetal long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, including heterozygosity for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation, nor fatty acid ,-oxidation defects in general are a major risk factor for HELLP syndrome in Austria. [source] | |||||||||||||