Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1991 Jan
PMID:Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis. 167 40

The Gunn rat, which is deficient in the UDP-glucuronosyltransferase for bilirubin, promptly excreted polar conjugates of the dimethyl ester of bilirubin in bile after intravenous infusion of this ester. The conjugates proved to be monoglutathione thioether adducts of the vinyl groups of the parent tetrapyrrole. High performance liquid chromatographic analysis of the conjugates as their dipyrrolic azosulfanilates demonstrated that only one of the dipyrroles of each tetrapyrrole was conjugated. The nonconjugated dipyrrole eluted as either the methyl endo- or exovinyl azodipyrrole. The amino acid composition of the pigments was consistent with that of a monoglutathione conjugate. NMR spectroscopy of the two major pigments demonstrated the loss of the proton signals of the C-18 vinyl group, indicating it to be the site of conjugation. Cation fast atomic bombardment tandem mass spectrometry demonstrated a molecular ion, [M + H]+, of m/z 937, which fragmented with a loss of 307 atomic mass units, consistent with glutathione. A molecular ion of m/z 807 was observed for the conjugate treated with gamma-glutamyltranspeptidase, consistent with the loss of glutamate. The mass spectrometry data indicated that the conjugates also contained a functional group whose mass was equivalent to hydroxyl, suggesting initial formation of an epoxide, which then reacts with glutathione. Pretreatment of the rat with 2,3,7,8-tetrachlorodibenzo-p-dioxin to induce cytochrome P-450 resulted in a 6-fold increase of the biliary excretion of the glutathione conjugates. Such induction also resulted in the excretion of a glutathione conjugate of bilirubin itself.
Mol Pharmacol 1991 Oct
PMID:Identification of glutathione conjugates of the dimethyl ester of bilirubin in the bile of Gunn rats. 168 18

The significance of glucose-6-phosphatase (G6P) expression by bile duct-like cells proliferating during hepatocarcinogenesis in the histogenesis of hepatocellular carcinoma is not clear. To this end, we measured the histochemical and biochemical activity of G6P in normal rat liver, and in rat livers in which bile duct-like proliferation was induced by either hyperplastic (bile duct ligation for 14 days or feeding alpha-naphthylisothiocyanate for 28 days) or neoplastic (feeding a choline-devoid diet containing 0.1% ethionine for 60 days) regimens. In normal, hyperplastic, and preneoplastic livers, G6P histochemical activity was confined to the hepatocytes; proliferated bile duct-like cells, like normal bile ducts, did not display visible G6P staining. When the enzyme activity was determined biochemically, however, hydrolysis of glucose-6-phosphate was observed in both parenchymal and nonparenchymal liver cells isolated from all experimental animals. In elutriated nonparenchymal fractions, G6P activity was directly proportional to the number of cells positive for gamma-glutamyl transpeptidase and cytokeratin no. 19 (markers of bile duct cells) and inversely proportional to the number of cells positive for vimentin (marker of mesenchymal cells). These results indicate that, while by light microscopy hepatic G6P histochemical activity is detectable only in the hepatocytes, the biochemical activity is also expressed in proliferating bile duct-like cells. However, the nonparenchymal activity is observed during both neoplastic and hyperplastic liver growth, thus indicating that the presence of this enzyme in bile duct-like cells proliferating during hepatocarcinogenesis should not necessarily be construed as supporting their stem cell nature nor their neoplastic commitment.
Virchows Arch B Cell Pathol Incl Mol Pathol 1991
PMID:Distribution of glucose-6-phosphatase activity in normal, hyperplastic, and preneoplastic rat liver. 168 20

The cellular origin of estrogen-induced kidney tumors in male Syrian hamsters has been repeatedly the subject of controversy. Several authors have proposed that the tumors arise from proximal tubules, from a combination of tubular and interstitial stromal cells, or solely from interstitial cells. Because of the model character of this tumor for hormone-associated cancer, it was further investigated in this study with respect to morphology, enzyme and intermediate filament pattern, the expression of alpha-smooth muscle actin and the extracellular matrix proteins fibronectin and tenascin. These analyses were carried out with early and late tumors as well as metastases to determine possible changes in expression of biochemical parameters during the development and progression of this neoplasm. The enzyme histochemical and intermediate filament patterns were usually the same as those described previously for proliferative foci and early tumors, i.e. highly elevated activities of glucose-6-phosphate dehydrogenase, adenylate cyclase and alkaline phosphatase, a lack of glucose-6-phosphatase and gamma-glutamyltransferase and coexpression of vimentin and desmin, alpha-smooth muscle actin could not be detected in early lesions. In five of 24 advanced tumors inclusions of kidney tubules were found which showed various degrees of alteration in their morphology and enzyme histochemical pattern, but were often directly connected with tubular segments of normal appearance outside the tumor. Like the normal tubules, the enclosed tubular segments were strongly positive for cytokeratin but never expressed vimentin or desmin. Among the 24 tumors studied, two contained cysts which expressed cytokeratin and sometimes also vimentin but not desmin. The enzyme histochemistry of the cells lining the cysts was similar to that of the surrounding tumor mass, except adenylate cyclase was lacking and alkaline phosphatase was not uniformly distributed. In tumors containing cytokeratin-positive cysts, there often were cytokeratin-positive, vimentin-negative and desmin-negative tumor formations in close contact to these cysts. With the exception of cyst formation, the pattern of metastases were identical to that of the primary tumors. All large tumors and the main component of the metastases expressed vimentin, desmin and fibronectin. Mesothelia surrounding metastatic tumor complexes were positive for vimentin, desmin, alpha-smooth muscle actin, fibronectin, cytokeratin and tenascin. It was concluded from these and previous observations on early stages of tumor development that the estrogen-induced hamster kidney tumor originates from mesenchymal interstitial cells (probably pericytes) which may rarely acquire an epithelial phenotype by metaplastic transformation during tumor progression.
Virchows Arch B Cell Pathol Incl Mol Pathol 1991
PMID:Changes in the cellular phenotype and extracellular matrix during progression of estrogen-induced mesenchymal kidney tumors in Syrian hamsters. 171 81

A high frequency of point mutations at codon 12 of the Ki-ras gene has previously been reported for rat kidney mesenchymal tumors induced by methylating N-nitroso compounds. In this study, we analyzed renal tumors with divergent histogenesis, i.e., mesenchymal tumors (sarcomas), cortical epithelial tumors (carcinomas), and embryonal tumors (nephroblastomas). Renal mesenchymal tumors and carcinomas were induced in juvenile or young adult Wistar rats by a single dose of N-nitrosodimethylamine (NDMA) while nephroblastomas were induced in Nb hooded rats by a single transplacental dose of N-nitrosoethylurea (NEU). Nephroblastomas developing spontaneously in WAB/Not rats were also examined. Amplification of Ki-ras sequences from formalin-fixed, paraffin-embedded tissue by the polymerase chain reaction was followed by direct DNA sequencing. GGT----GAT point mutations at codon 12 of the Ki-ras gene were found in 9 of 12 (75%) renal mesenchymal tumors and in 9 of 12 (75%) cortical epithelial tumors induced by NDMA. Even higher incidences were observed in nephroblastomas (8/8; 100%) induced by NEU and in spontaneous nephroblastomas (10/11; 91%). These results indicate that Ki-ras mutations are frequent events during the development of kidney tumors irrespective of their histogenesis and suggest that they may play an important role in renal carcinogenesis in rats. These data further indicate that mutational activation of Ki-ras proto-oncogenes in carcinogen-induced rat kidney tumors occurs in a tissue-specific, rather than cell-specific, manner.
Mol Carcinog 1991
PMID:Ki-ras mutations in spontaneous and chemically induced renal tumors of the rat. 179 84

H69AR is a multidrug-resistant small cell lung cancer cell line derived from a drug-sensitive cell line, H69, by selection in doxorubicin. It is cross-resistant to a wide variety of natural product-type antineoplastic agents but does not overexpress P-glycoprotein. In the present study, the levels of GSH and GSH-related enzymes in the H69AR cell line were determined and compared with those found in H69 cells. Unlike other drug-resistant cell lines, GSH levels were diminished 6-fold in H69AR cells (0.67 +/- 0.28 microgram/mg of protein), compared with H69 cells (4.23 +/- 1.17 micrograms/mg of protein) (p less than 0.01). This unusually low level of GSH may explain the pronounced collateral sensitivity of H69AR cells to buthionine sulfoximine (BSO), an inhibitor of the rate-limiting enzyme in GSH biosynthesis (ID50 of 4.4 microM BSO for H69AR cells versus ID50 of 300 microM BSO for H69 cells). BSO did not enhance doxorubicin cytotoxicity in the H69AR cell line, despite further depletion of GSH. GSH-reductase (EC 1.6.4.2) activity was elevated 2-fold in H69AR cells, compared with sensitive H69 cells (75.34 +/- 14.94 versus 38.62 +/- 5.06 nmol of NADPH/min/mg of protein) (p less than 0.05). Both selenium-dependent and -independent GSH-peroxidase (EC 1.11.1.9) activities were unchanged in the resistant H69AR cell line, compared with its parent cell line. gamma-Glutamyl transpeptidase (EC 2.3.2.2) activity was 5-fold elevated in H69AR cells, compared with H69 cells (2.50 +/- 0.44 versus 0.46 +/- 0.21 nmol of p-nitroaniline/min/mg of protein) (p less than 0.01), whereas GSH-S-transferase (EC 2.5.1.18) activity was 10-fold higher (201.98 +/- 43.62 versus 19.77 +/- 1.72 nmol of 1-chloro-2,4-dinitrobenzene/min/mg of protein in H69AR and H69 cells, respectively) (p less than 0.01). The GSH-S-transferases from both cell lines were purified by affinity chromatography and immunoblot analysis identified the GSH-S-transferases as belonging to the anionic pi class. GSH-S-transferases from the mu or alpha classes were not detectable in either cell line. In conclusion, marked differences in GSH levels and the activities of three of four GSH-related enzymes were observed between the multidrug-resistant H69AR cell line and its parent cell line. Further study is required to determine whether these changes are causally related to the development of drug resistance in this model system.
Mol Pharmacol 1990 Feb
PMID:Alterations in glutathione and glutathione-related enzymes in a multidrug-resistant small cell lung cancer cell line. 196 21

Several lines of evidence suggest that the renal-specific toxicity of quinol-linked GSH conjugates is probably a result of their metabolism by gamma-glutamyl transpeptidase and selective accumulation by proximal tubular cells. Transport of the resultant quinol-cysteine and/or cystein-S-ylglycine conjugate followed by oxidation to the quinone may be important steps in the mechanism of toxicity of these compounds. Factors modulating the intracellular and/or intralumenal concentration of the cystein-S-yl and cystein-S-ylglycine conjugate will, therefore, be important determinants of toxicity. We have now studied the gamma-glutamyl transpeptidase-mediated metabolism of 2-bromo-3-(glutathion-S-yl)hydroquinone. The product of this reaction, 2-bromo-3-(cystein-S-ylglycyl)hydroquinone, undergoes an intramolecular cyclization to yield a 1,4-benzothiazine derivative that retains the glycine residue. A similar cyclization reaction occurs with 2-bromo-3-(cystein-S-yl)hydroquinone, which is unstable in aqueous solutions and undergoes a pH-dependent rearrangement that requires initial oxidation to the quinone. UV spectroscopy revealed that, at neutral pH, further reaction results in the formation of a chromophore, consistent with 1,4-benzothiazine formation. This product arises via cyclization of the cysteine residue via an intramolecular 1,4 Michael addition. Further reaction results in the precipitation of a pigment that exhibits properties of a pH indicator. The pigment undergoes a marked pH-dependent bathochromic shift (approximately 100 nm); it is red in alkali (lambda max, 480 nm) and violet in acid (lambda max, 578 nm). These properties are similar to those of the trichochrome polymers that are formed during melanin biosynthesis from S-(3,4-dihydroxyphenylalanine)-L-cysteine. Because the intramolecular cyclization reactions remove the reactive quinone moiety from the molecules, they may be regarded as detoxication reactions. 1,4-Benzothiazine formation represents a novel pathway that diverges from the usual route of mercapturic acid synthesis and may represent previously unrecognized and important products of quinone metabolism in vivo.
Mol Pharmacol 1990 Jul
PMID:Oxidative cyclization, 1,4-benzothiazine formation and dimerization of 2-bromo-3-(glutathion-S-yl)hydroquinone. 197 24

The molecular genetics of human endometrial carcinoma have yet to be defined to any significant extent. Cell lines from 11 endometrial carcinomas were examined for alterations in proto-oncogenes that might predictably be present, based on existing data from the better-characterized human carcinomas of the uterine cervix, ovary, and breast. Codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes were examined for possible point mutations, and the c-erbB2/neu, c-myc, and epidermal growth factor receptor (EGFR) genes were examined for amplification or overexpression. Ras mutations were found in seven of 11 (64%) tumors, including three in codon 61 of Ha-ras (CAG----CAT) and four in codon 12 of Ki-ras (GGT----GAT in two and GGT----GTT in two). No evidence was found for amplification or overexpression of the c-erbB2 or EGFR genes in any tumor. One tumor contained amplified c-myc sequences and exhibited relative overexpression of c-myc. These data suggest that the amplification or overexpression of several proto-oncogenes frequently observed in other human gynecologic and breast tumors are not prevalent in endometrial carcinoma and that ras gene mutations are relatively common in this tumor type.
Mol Carcinog 1991
PMID:Analysis of oncogene alterations in human endometrial carcinoma: prevalence of ras mutations. 206 24

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain activated Ha-ras oncogenes capable of inducing morphologic and tumorigenic transformation of NIH 3T3 cells. In this study, we analyzed human primary squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) occurring on sun-exposed body sites for mutations in codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction, followed by dot-blot hybridization to synthetic oligonucleotide probes designed to detect single base-pair mutations. In addition to the primary human skin cancers, we also analyzed Ha-ras-positive NIH 3T3 transformants for mutations in the Ha-ras oncogene. The results indicated that all three NIH 3T3 transformants, 11 of 24 (46%) SCCs, and 5 of 16 (31%) BCCs contained mutations at the second position of Ha-ras codon 12 (GGC----GTC), predicting a glycine-to-valine amino acid substitution, whereas only 1 of 40 skin cancers (an SCC) displayed a mutation in the first position of Ki-ras codon 12 (GGT----AGT), predicting a glycine-to-serine amino acid change. In addition, three of the SCCs contained highly amplified copies of the N-ras oncogene in their genomic DNA. Interestingly, two of the SCCs containing amplified N-ras sequences also had G----T mutations in codon 12 of the Ha-ras oncogene. These studies demonstrate that mutations in codon 12 of the Ha-ras oncogene occurred at a high frequency in human skin cancers originating on sun-exposed body sites, whereas mutation in codon 12 of Ki-ras or amplification of N-ras occurred at a low frequency. Since the mutations in the Ha-ras and Ki-ras oncogenes were located opposite potential pyrimidine dimer sites (C-C), it is likely that these mutations were induced by ultraviolet radiation present in sunlight.
Mol Carcinog 1991
PMID:Ras gene mutation and amplification in human nonmelanoma skin cancers. 206 25

We have used the polymerase chain reaction and dideoxynucleotide sequencing to amplify and sequence exons 1 and 2 of the c-Ki-ras proto-oncogene from the normal Syrian golden hamster. Similar methods were employed to screen for the presence of point mutations in the c-Ki-ras oncogene in primary hamster pancreatic ductal adenocarcinomas (PDC) induced by N-nitrosobis(2-oxopropyl)amine (BOP). A GGT to GAT point mutation was detected in codon 12 of the c-Ki-ras gene in 10 primary hamster PDCs. This same point mutation was present in two nonclonal cell lines, PC-1 and PC-1-0, established from tumors that were produced in hamsters by subcutaneously implanting a preparation of minced BOP-induced PDC. Two clonal cell lines, Cl-3 and Cl-7, were cloned from the PC-1 cell line, and these cell lines also carried the GAT point mutation at codon 12. This point mutation was the same as that detected in greater than 75% of adenocarcinomas from the human exocrine pancreas. Thus, our findings provide further validation for the use of the BOP-induced hamster PDC model as a relevant experimental model for human pancreas cancer: not only did the hamster pancreatic ductal adenocarcinomas closely resemble their human counterpart in histopathological morphology and sequential development, but they also contained the same point mutation in codon 12 of the c-Ki-ras oncogene, as has been reported for human pancreatic adenocarcinomas.
Mol Carcinog 1990
PMID:Pancreatic ductal adenocarcinomas induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine contain a c-Ki-ras oncogene with a point-mutated codon 12. 217 32


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