Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Genomic DNA for genetic analyses has traditionally been derived from blood samples. With the availability of PCR techniques requiring only minute amounts of DNA and the current demand for high-volume testing, a less invasive, simpler to perform, and cheaper method to obtain DNA is desirable. We developed a method to obtain high-quality genomic DNA from buccal cells that has high acceptability and allows for a large number of PCR assays from a single sample. Sixty subjects vigorously swished 10 ml of undiluted commercial mouthwash in the mouth for 60 s and expelled the liquid into a collection container. DNA was isolated from the buccal cells with a rapid method using proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. Electrophoretic analysis of the extracted DNA showed detectable levels of high molecular weight genomic DNA in all samples. The DNA yields ranged from 0.2 to 134.0 microg, for an average of 49.7 microg. Using these samples, all 60 subjects were successfully genotyped by PCR-based assays for polymorphisms in the CYP1A1 (MspI and exon 7), CYP2E1 (RsaI), GSTM1, GSTT1, and NQO1 genes, confirming that the quality of DNA isolated from mouthwash samples was sufficient to reliably support PCR amplification. Storage of the (unprocessed) specimens at room temperature or at 37 degrees C for 1 week (temperature conditions that may be encountered when mailing samples) or at -20 degrees C for at least 6 months did not affect the DNA yield or ability to PCR amplify the samples. The results suggest that this mouthwash procedure may be suitable for large community-based studies of genetic susceptibility to disease in which samples can be collected by the participants themselves, mailed back to the study center, and stored for months prior to DNA analysis.
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PMID:A simple mouthwash method for obtaining genomic DNA in molecular epidemiological studies. 971 25

This study evaluates the influence of genetic polymorphism at GSTM1, GSTM3 and GSTT1 gene loci on oral cancer risk among Indians habituated to the use of, smokeless tobacco, bidi or cigarette. DNA extracted from white blood cells of 297 cancer patients and 450 healthy controls by the proteinase K phenol-chloroform extraction procedure were analyzed by the polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) analyses. Lifetime tobacco exposure was evaluated as a risk factor in relation to the polymorphism at the GST gene loci using logistic regression analysis. There was no significant difference in the distribution of the GSTM3 and GSTT1 genotypes between oral cancer patients and controls. In contrast, a significant 3-fold increase in risk was seen for patients with the GSTM1 null genotype (age adjusted OR = 3.2, 95% CI 2.4-4.3). The impact of the GSTM1 null genotype on oral cancer risk was also analyzed in separate groups of individuals with different tobacco habits. The odds ratio associated with the GSTM1 null genotype was 3.7 (95% CI 2.0-7.1) in tobacco chewers, 3.7 (5% CI 1.3-7.9) in bidi smokers and 5.7 (95% CI 2.0-16.3) in cigarette smokers. Furthermore, increased lifetime exposure to chewing tobacco appeared to be associated with a 2-fold increase in oral cancer risk in GSTM1 null individuals. The results suggest that the GSTM1 null genotype is a risk factor for development of oral cancer among Indian tobacco habitues.
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PMID:Polymorphism at GSTM1, GSTM3 and GSTT1 gene loci and susceptibility to oral cancer in an Indian population. 1201 53

The glutathione S-transferase (GST) family of enzymes has a vital role in phase II of biotransformation of environmental carcinogens, pollutants, drugs and other xenobiotics. GSTs are polymorphic, with the type and frequency of polymorphism being ethnic dependent. Polymorphisms in GST genes have been shown to be associated with susceptibility to disease and disease outcome. We determined the frequencies of GSTM1, GSTT1 and GSTP1 polymorphisms in 591 volunteers who had been residents of Rio de Janeiro for at least six months. Blood was collected and DNA extracted by proteinase K/SDS digestion. Information about social habits and health problems was also recorded. GSTM1 and GSTT1 polymorphisms were analyzed by a PCR-Multiplex procedure, whereas GSTP1 polymorphism was analyzed by PCR-RFLP. We found that 42.1% (48.9% of whites and 34.2% of non-whites) of the individuals had the GSTM1 null genotype, whereas 25.4% (25.1% of whites and 25.7% of non-whites) had the GSTT1 null genotype. The genotypic distribution of GSTP1 was 49.7% I/I, 38.1% I/V, and 12.2% V/V, whereas the allelic frequencies were 0.69 for the Ile allele, and 0.31 for the Val allele. The frequencies of GST polymorphisms in this Brazilian population were found to be different from those observed in other populations, particularly of other South American countries.
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PMID:Frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms in a Brazilian population. 1496 30

Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and the occurrence of tumors in different species, the mechanism of tumor induction by DCM metabolites produced through the GST pathway remains unclear. In this study a V79 cell line stably transfected with the murine GST theta 1 gene (mGSTT1) was compared to the parent cell line (MZ) to determine how the construct affects DCM metabolism and the sensitivity of the cell line to DNA damage and cytotoxicity. V79 cells were treated with DCM (2.5-10mM) or formaldehyde (150-600muM) for 2h. Also, formaldehyde produced by V79 cytosol metabolism of DCM was measured spectrophotometrically. DNA damage and DNA-protein crosslinks were measured by the standard and proteinase K-modified alkaline single cell gel electrophoresis (SCG) assays. Cytotoxicity was assessed by trypan blue stain exclusion, the Live/Dead((R)) cell viability/cytotoxicity kit for animal cells, and the neutral red assay. After DCM treatment a significant concentration-dependent increase in tail moment in the V79 MZ cells was observed compared to a significant concentration-dependent decrease in tail moment in the V79 mGSTT1 cells. Post-incubation with proteinase K significantly increased DNA migrations in DCM-treated V79 mGSTT1 cells. DCM formed significantly higher levels of formaldehyde in the cytosol of the V79 mGSTT1 cells than in the cytosol of the V79 MZ cells. Results using the cytotoxicity assays were comparable using the trypan blue and Live/Dead((R)) assays, neither showing a difference in response between the two cell lines when exposed to either formaldehyde or DCM. These results indicate that V79 mGSTT1 can metabolize DCM to a genotoxic and cytotoxic metabolite, which is likely formaldehyde. This is the first time that the magnitude of the GSTT1 effect can be observed in mammalian cells without confounding caused by using cells with different genetic backgrounds.
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PMID:Induction of DNA-protein crosslinks by dichloromethane in a V79 cell line transfected with the murine glutathione-S-transferase theta 1 gene. 1676 33

Colorectal cancer is the second most frequent cause of death among malignant diseases. The mortality of head and neck cancer in Hungary increased by 265 percent in the last thirty years. Both malignancies belong to the most current public health problems in Hungary. The influence of two allelic polymorphisms of GSTM1 and GSTT1, and that of p53 gene codon 72 on colon cancer was investigated. In case of head and neck cancer the effects of the Arg194Trp and Arg399Gln polymorphisms of XRCC1 gene were analyzed. Intraoperative removed tissue samples were processed and cancer free human samples were used as matched controls. The formalin fixed samples were deparaffinized and digested with proteinase K. Genotyping was performed by PCR amplification, and in case of head and neck cancer a PCR-RFLP method was applied. No significant difference was found between tumor patients and controls in the investigated polymorphisms. A significant difference in survival was found between the GSTM1 and p53 gene variants in Dukes'B stage colorectal patients. The survival difference among the XRCC1 194 alleles by head and neck patients in clinical stage III proved to be also significant. The complex analysis of this type of genetic variants may be the future way of the personal risk assessment and the real chance for personal therapy.
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PMID:[Genetic polymorphism in patients with colorectal and with head and neck cancer]. 1964 19

The main aim of this study was to evaluate genotoxic effects of pesticides in association with glutathione S-transferase (GST) polymorphism. To achieve this aim, DNA damage and the genotypes of the GSTM1 and GSTT1 genes were studied from blood lymphocytes of pesticide-exposed and unexposed (control) agricultural workers of the Punjab region of northwestern India. The blood samples were collected from 40 exposed and 27 unexposed subjects from the Kakrala and Sanour villages of Patiala district. DNA damage was evaluated by using an alkaline comet assay. The analysis of the comets was done through visual scoring and image analysis software (Tritek's CometScore). Damage Index (DI), Damage Frequency (DF) (calculated by visual scoring method), and % DNA in tail (measured by image analysis software) were considered for assessing DNA damage. The DNA extraction from blood cells was done using proteinase K and the phenol-chloroform method, and genotyping of GSTM1 and GSTT1 was done using multiplex PCR. It was found that all the pesticide-exposed subjects showed higher DI, DF, and % DNA in tail in comparison to the controls. The statistical comparison of DNA damage between the exposed group and unexposed group revealed highly significant differences (p < 0.05; Mann-Whitney U-test). In addition, the GSTT1 gene deletion and simultaneous deletions of GSTM1 and GSTT1 genes in increasing DNA damage were observed in the exposed group.
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PMID:Association of GSTM1 and GSTT1 gene deletions with susceptibility to DNA damage in the pesticide-exposed workers of Punjab. 2037 Apr 84