Distinctive structure of the human GSTM3 gene-inverted orientation relative to the mu class glutathione transferase gene cluster

The goals of this study were 1) to compare the effects of transforming growth factor-beta (TGF-beta) and parathyroid hormone-related protein (PTHrP) on mouse blastocyst attachment and outgrowth in vitro, 2) to determine whether TGF-beta acts through a mechanism involving PTHrP, 3) to examine effects of PTHrP on preimplantation mouse embryo development, and 4) to determine the pattern of expression of PTHrP protein in the uterus of the mouse during early gestation. In the first set of experiments, hatched blastocysts were placed in fibronectin-coated wells. Cultures were treated with PTHrP or TGF-beta1 and assessed at 24, 48, and 72 h for attachment and surface area of blastocyst outgrowth. Results showed that both PTHrP and TGF-beta1 increased blastocyst outgrowth significantly. A PTHrP-neutralizing antibody blocked the stimulatory effect of both PTHrP and TGF-beta1, suggesting that TGF-beta1 acts to increase endogenous production of PTHrP by the blastocyst. Immunoassay of conditioned medium from blastocysts treated with either TGF-beta1 or PTHrP 1-34 confirmed a 3- to 4-fold increase in levels of PTHrP 1-141. In the second series of experiments, pronuclear zygotes were cultured in various concentrations of PTHrP for 96 h. Blastocysts then were subjected to differential fluorescent staining of inner cell mass and trophectoderm cells. Treatment of mouse embryos with the various concentrations of PTHrP altered neither the number developing to the blastocyst stage nor the number of inner cell mass or trophectoderm cells in the resulting blastocysts. In the third experiment, pregnant mice were killed at Days 3, 4, 5, 6, and 7 of gestation, and uterine horns were processed for immunohistochemistry. Uterine sections were stained with antibodies to PTHrP, desmin, and laminin. On Days 3, 4, and 5, uterine luminal and glandular epithelial cells stained intensely for PTHrP, while stromal cells were negative. By Days 6 and 7, decidualized stromal cells stained positively for PTHrP, desmin, and laminin. These results support the hypothesis that TGF-beta and PTHrP play an important role in the process of implantation.     

Phenotyping of glutathione S-transferase M1 in the Estonian population by ELISA using GSTM1a and GSTM1b specific monoclonal antibodies

We examined the effects of stimulation on either postnatal days 1-7 or 21-27 on passive avoidance reaction (PAR) of young rats. Animals received tactile or visual stimulation for 10 min each day, and were trained on postnatal day 28 in a step-through apparatus using a footshock of 0.75 mA for 2 s. Retention was tested on five consecutive days beginning on day 29. Memory retention was measured for each rat 24, 48, 72, 96 and 120 h after the acquisition trial. Step-through latencies to enter the dark compartment, time spent in the illuminated compartment and number of crossings of the light beam were recorded up to 200 s. Rats that received tactile or visual stimulation during the 4th postnatal week displayed significantly lower PAR latencies, a shorter stay in the illuminated compartment and a higher number of crossings of the light beam compared to rats treated during the 1st postnatal week. The untreated control group showed a rapid decline of PAR latencies. All experimental groups remained in the illuminated compartment longer and showed PAR latencies well above those of the control group. The differences became more pronounced when visual stimulation in the first postnatal week was used. The number of crossings of the light beam was significantly reduced by the treatment, with the exception of the experimental group stimulated visually in the 4th week. The behavioural changes induced by tactile or visual stimulation have a long-lasting effect in coping with a stressful task.     

Solution structure of the carboxyl terminus of a human class Mu glutathione S-transferase: NMR assignment strategies in large proteins

Strategies to obtain the NMR assignments for the HN, N, CO, Calpha and Cbeta resonance frequencies for the human class mu glutathione-S-transferase GSTM2-2 are reported. These assignments were obtained with deuterated protein using a combination of scalar and dipolar connectivities and various specific labeling schemes. The large size of this protein (55 kDa, homodimer) necessitated the development of a novel pulse sequence and specific labeling strategies. These aided in the identification of residue type and were essential components in determining sequence specific assignments. These assignments were utilized in this study to characterize the structure and dynamics of the carboxy-terminal residues in the unliganded protein. Previous crystallographic studies of this enzyme in complex with glutathione suggested that this region may be disordered, and that this disorder may be essential for catalysis. Furthermore, in the related class alpha protein extensive changes in conformation of the C terminus are observed upon ligand binding. On the basis of the results presented here, the time-averaged conformation of the carboxyl terminus of unliganded GSTM2-2 is similar to that seen in the crystal structure. NOE patterns and 1H-15N heteronuclear nuclear Overhauser enhancements suggest that this region of the enzyme does not undergo motion on a rapid time scale.     

Role of glutathione S-transferase GSTM1, GSTM3, GSTP1 and GSTT1 genotypes in modulating susceptibility to smoking-related lung cancer

The prevalence and risk factors for nosocomial lower respiratory tract infections (LRTI) in Germany were determined as part of a national survey on nosocomial infections. The study included 14,966 patients in 72 representatively selected hospitals with departments of general medicine, surgery, obstetrics, gynecology, and intensive care units (ICU). Surveillance was carried out by four previously validated medical doctors who strictly applied the CDC-criteria for diagnosis of nosocomial infections. The overall prevalence of hospital-acquired LRTI was 0.72% with the highest rate in hospitals with more than 600 beds (1.08%) and among the patients on intensive care units (9.00%). Ventilator-associated pneumonia rates were highest in patients on ICUs (13.27). Polytrauma, impaired consciousness, chronic airway disease, prior surgery, and cardiovascular disease were significantly related to the occurrence of nosocomial LRTI. P. aeruginosa was the predominant organism causing nosocomial LRTI. Nosocomial LRTI remain a problem mainly on ICUs. Patients at risk should be monitored with extra care.     

Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia

Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents. The pi-class GST has been associated with preneoplastic and neoplastic changes. Recently, it has been reported that regulatory sequences near the GSTP1 gene, which encodes the human pi-class GST, are commonly hypermethylated in prostatic carcinomas. In the present study, we studied more than 300 primary human tumors originating in other organs for aberrant methylation of GSTP1 using methylation-specific PCR. GSTP1 hypermethylation was most frequent in breast and renal carcinoma, showing aberrant methylation in 30 and 20% of the cases, respectively. Other tumor types showed promoter methylation only rarely or not at all. Hypermethylation of GSTP1 was associated with loss of expression demonstrated by immunohistochemistry. Our results suggest that aberrant methylation of GSTP1 may contribute to the carcinogenetic process in breast and renal carcinomas.     

Structure and organization of the human alpha class glutathione S-transferase genes and related pseudogenes

We have isolated and characterized genomic DNA encoding several human Alpha class glutathione S-transferase genes and pseudogenes. All the genes are composed of seven exons with boundaries identical to those of the Alpha class genes in rats. The GSTA1 gene is approximately 12 kb in length and is closely flanked by other Alpha class gene sequences. The complete sequence of the 1.7-kb intergenic region between exon 7 of an upstream pseudogene and exon 1 of the GSTA1 gene has been determined. An additional gene that encodes an uncharacterized Alpha class glutathione S-transferase has been identified. The protein derived from this gene would have 19 amino acid substitutions compared with the GSTA1 isoenzyme. Several pseudogenes with single-base and/or complete exon deletions have been identified, but no reverse-transcribed pseudogenes have been detected. The occurrence of multiple genes and pseudogenes on a single fragment of cloned genomic DNA and the prior identification of a single chromosomal region (6p12) of hybridization (Board and Webb, 1987, Proc. Natl. Acad. Sci. USA 84:2377-2381) suggest that all the Alpha class genes are members of a closely linked gene family that has evolved by duplication and gene conversion events.     

Association of glutathione S-transferase GSTM1 and GSTT1 null genotypes with clinical outcome in epithelial ovarian cancer

Epithelial ovarian cancer is generally associated with a poor outcome, although the mechanisms that determine survival and progression-free interval (PFI) are unclear. Data from ovarian tumors showing associations between (a) null genotypes at the glutathione S-transferase GSTM1 and GSTT1 loci and expression of p53 protein and (b) outcome and expression of p53 suggest that polymorphism at these loci is a factor determining outcome. Accordingly, we have studied the association between the GSTM1 null and GSTT1 null genotypes and survival and PFI in 148 women with epithelial ovarian cancer. Although we did not find an association between individual genotypes and outcome, women with both GSTM1 null and GSTT1 null genotypes demonstrated poorer survival (P = 0.001) and reduced PFI (P = 0.003). Thus, no cases with both these genotypes survived past 42 months postdiagnosis. In contrast, 43% of the women without this combination survived beyond this time. Because response to chemotherapy is a major factor determining outcome in ovarian cancer, we also examined the data for associations between the glutathione S-transferase genotypes and response to such treatment. Thus, in 78 patients treated with chemotherapy, the combination of GSTM1 null and GSTT1 null was associated with unresponsiveness to primary chemotherapy (P = 0.004); none of the eight patients with both these genotypes responded, compared with 38 of 70 (54%) of patients with other genotype combinations. The effect of the combination of genotypes on survival and PFI was lost in a multivariate model that included response to chemotherapy as a confounding factor. This suggests that the combination of GSTM1 null/GSTT1 null is associated with outcome because of its influence on response to chemotherapy. These preliminary findings may provide a basis for the selection of patients for treatment with chemotherapeutic agents.     

Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes

The crystal structure of human alpha class glutathione transferase A1-1 has been determined and refined to a resolution of 2.6 A. There are two copies of the dimeric enzyme in the asymmetric unit. Each monomer is built from two domains. A bound inhibitor, S-benzyl-glutathione, is primarily associated with one of these domains via a network of hydrogen bonds and salt-links. In particular, the sulphur atom of the inhibitor forms a hydrogen bond to the hydroxyl group of Tyr9 and the guanido group of Arg15. The benzyl group of the inhibitor is completely buried in a hydrophobic pocket. The structure shows an overall similarity to the mu and pi class enzymes particularly in the glutathione-binding domain". The main difference concerns the extended C terminus of the alpha class enzyme which forms an extra alpha-helix that blocks one entrance to the active site and makes up part of the substrate binding site.     

Functions of His107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a

Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases. Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties. With 1-chloro-2, 4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pKa of kcat/Km from 7.8 for the Ser107-containing enzymes to 6.3 for the His107-containing enzymes. There is a parallel shift in the pKa for thiolate anion formation of enzyme-bound GSH. Y6F mutations have no effect on the pKa for these enzymes. Crystal structures of hGSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 A from the GSH thiol group. Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation. All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (Kd values of 1-2 x 10(-)6 M); however, KmGSH and Ki values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher. The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases. Consequences of His107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting.     

Polymorphic expression of the glutathione S-transferase P1 gene and its susceptibility to Barrett's esophagus and esophageal carcinoma

Factors determining individual susceptibility to esophageal cancer or premalignant Barrett's epithelium are still largely unclear. An imbalance between phase I drug metabolism [e.g., cytochrome P450 (CYP)] and phase II detoxification [e.g., glutathione S-transferase (GST)] may contribute to the development of these diseases. Polymorphic variants in the CYP1A1 gene were described leading to increased levels of bioactive compounds, whereas polymorphisms in GST genes often resulted in impaired detoxification. We studied the frequencies of polymorphic variants in CYP1A1, GSTP1, GSTT1, and GSTM1 genes in 98 patients with Barrett's epithelium and 34 patients with esophageal cancer. The results were compared with those obtained from 247 healthy blood donors. DNA was extracted, and PCR-RFLP methods were used to detect genetic polymorphisms. Chi2 analysis, Spearman rank correlation, and Wilcoxon rank sum tests were used for statistical evaluation. Polymorphisms in CYP1A1, GSTM1, and GSTT1 occurred at an equal frequency in patients and controls. Occurrence of the polymorphic GSTP1b variant in the GSTP1 gene resulted in a significantly lower GST enzyme activity (P < 0.05), and GSTP1b was found significantly more often in patients with Barrett's epithelium (70%; P < 0.001) and patients with esophageal adenocarcinoma (76%; P = 0.005), as compared to healthy blood donors (41%). In conclusion, presence of the GSTP1b allele leads to lower GST enzyme activity levels and, consequently, impaired detoxification. This most important esophageal GST isoform may, therefore, contribute to the development of Barrett's epithelium and adenocarcinoma.     

Structural studies of a human pi class glutathione S-transferase. Photoaffinity labeling of the active site and target size analysis

The present study analysed gubernaculum development in mice that had been induced, through transgenesis, to express human anti-Müllerian hormone (h-AMH) throughout prenatal life. Growth and differentiation of the gubernacular primordia were assessed through the analysis of serial, transverse or sagittal, histological sections of the lower abdomen. Transgenic males and females expressed biologically active amounts of h-AMH as measured by sensitive and specific ELISA and evidenced through the regression, in females, of Müllerian ducts after day 13 of prenatal life. Gubernacular primordia became distinguishable at the same age in control and transgenic male and female fetuses on day 12 after coitus. In both groups gubernacular cords (inguinal folds of the genital mesenteries) increased in length more in females than in males while gubernacular cones showed larger growth in males. h-AMH thus appeared not to affect the sexually dimorphic pattern of growth and development of these structures. Growth and differentiation of the gubernacular primordia was further examined in 18-day-old control and h-AMH transgenic fetuses that had been exposed to testosterone propionate injected into their mothers on days 12 and 14 of pregnancy. Testosterone treatment affected, to a minor extent, the growth of the female gubernacular cords: these were reduced in length (but had a larger surface area) compared with controls. The gubernacular cones were slightly increased in length but male-like differentiation of the tissues of the cones into a muscular and mesenchymal component was not noticed to any extent. The observations thus add experimental support to the contention that AMH, even in combination with testosterone, is not effective in establishing the male pattern of gubernacular primordia development.     

Characterization of monoclonal antibodies to the 26-kDa glutathione S-transferase of Schistosoma japonicum

Six monoclonal antibodies (MAbs) were raised in mice against the 26-kDa glutathione S-transferase (GST) of the parasite Schistosoma japonicum. These MAbs were originally selected for their specific binding to the recombinant GST (r-GST) generated in E. coli by an enzyme-linked immunosorbent assay. A further study demonstrated that all these MAbs bound to plate-coated GST affinity-purified from the parasite Schistosoma japonicum. However, in Western blotting analysis only a single monoclonal antibody (MAb Y3D7) yielded positive binding. The binding of MAb Y3D7 on Western blotting was further characterized; specific binding was found on other GST fusion proteins and on the authentic 26-kDa GST but not the 28-kDa GST in the total soluble worm proteins from Schistosoma japonicum. Using protein-A-mediated immunoprecipitation, MAbs Y3D7 and Y5D5 precipitated r-GST while in parallel experiments the remaining MAbs did not generate r-GST precipitation. In an alternative co-precipitation experiment, r-GST was first bound to glutathione (GSH) Sepharose beads and subsequently tested for interaction with the MAbs. In this manner, all MAbs except MAb Y5D5 were co-precipitated with the complexes. Thus, these select MAbs readily reacted with GST although their binding characteristics were different. Because GST has been widely used in the generation of fusion proteins for various purposes and is a potential vaccine candidate in controlling schistosomiasis, these MAbs should prove valuable for their application to molecular biology and parasitology.     

Role of cellular glutathione and glutathione S-transferase in the expression of alkylating agent cytotoxicity in human breast cancer cells

Glutathione (GSH) and glutathione S-transferases (GSTs) play an important role in the protection of cells against toxic effects of many electrophilic drugs and chemicals. Modulation of cellular GSH and/or GST activity levels provides a potentially useful approach to sensitizing tumor cells to electrophilic anti-cancer drugs. In this study, we describe the interactions of four representative alkylating agents (AAs), melphalan, 4-hydroperoxy-cyclophosphamide (4HC), an an activated form of cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and cisplatin, with GSH and GST in the human breast cancer cell line MCF-7. Depletion of cellular GSH pools by approximately 80% by treatment of the cells with the GSH synthesis inhibitor buthionine sulfoximine (BSO) sensitized the tumor cells to each AA to a different extent, with dose-modifying factors of 2.39, 2.21, 1.64, and 1.27 observed for melphalan, 4HC, cisplatin, and BCNU, respectively. Treatment of the cells with the GST inhibitor ethacrynic acid (EA) failed to show any significant effects on the cytotoxicity of these AAs. However, EA did potentiate the cytotoxicity of melphalan when given in combination with BSO, an effect that may be due to a more complete depletion of cellular GSH levels by the combined modulator treatment. Following a 1-hr exposure to cytotoxic-equivalent concentrations of these AAs, GSH levels decreased substantially in the case of 4HC and BCNU, but increased by 30-50% in the case of cisplatin and melphalan. BSO pretreatment largely blocked this effect of cisplatin and melphalan on cellular GSH, while it further enhanced the GSH-depleting activity of both 4HC and BCNU. On the basis of these results, it is concluded that (a) GSH affects the cytotoxicity of different AAs to different extents, (b) basal GST expression in MCF-7 cells does not play a major role in AA metabolism, (c) EA can potentiate the enhancing effect of BSO on melphalan cytotoxicity in MCF-7 cells, and (d) depletion of cellular GSH by pretreatment with BCNU or cyclophosphamide may correspond to a useful strategy for enhancing the anti-tumor activity of other AAs given in a sequential combination.     

Characterization and sequence of the Escherichia coli panBCD gene cluster

A 4589 bp DNA segment containing the Escherichia coli panBCD gene cluster was sequenced, and found to contain 6 complete open reading frames. panB, panC, and panD were identified by subcloning and insertional mutagenesis. The orientation of panD was also confirmed by orientation-specific expression of asparate-1-decarboxylase. panB and panC lie adjacent to one another, but are separated from panD by orf3, which is oriented in the opposite direction. Interruptions in the remaining open reading frames did not affect growth on glucose-minimal medium. No significant similarity to sequences in databases was found for orf1 and orf2. Orf3 contained extensive similarity to reading frames defined by E. coli yjiP, yjiQ, yhgA, and yafD. The function of these amino acid sequences is as yet undefined.     

A subfamily 2 homo-dimeric glutathione S-transferase mYrs-mYrs of class theta in mouse liver cytosol

A homo-dimeric subfamily 2 glutathione (GSH) S-transferase (GST) mYrs-mYrs of the class theta was isolated from mouse liver cytosol and purified to homogeneity. The first 28 N-terminal amino acid sequence of the GST was completely identical to that of rat subfamily 2 GST Yrs-Yrs of the class theta. GST mYrs-mYrs cross-reacted with anti-rat GST Yrs-anti-sera but not with anti-sera raised against rat GSTs Ya-Ya (alpha), Yb1-Yb1 (mu), and Yp-Yp (pi) and represented more than 95% of the mouse liver cytosolic GST activity to scavenge the reactive sulfate ester 5-sulfoxymethylchrysene of the potent carcinogen 5-hydroxymethylchrysene. The mouse class theta GST had little activity toward 1-chloro-2,4-dinitrobenzene and was unretainable on GSH and an S-hexyl-GSH affinity columns. GST mYrs-mYrs had a much higher GSH peroxidase activity toward fatty acid hydroperoxides than did the other classes of mouse GSTs.     

Effects of glutathione S-transferases GSTM1 and GSTT1 genotypes on lung cancer risk in smokers

BACKGROUND: Railway and subway-associated electrical trauma is rare and typically involves high voltage (> 20,000) arc injuries. Not all rail systems utilize such high voltage. We report 16 cases of electrical trauma due to 600 V direct contact with subway 'third' rails. METHODS: A case series of injured patients presenting to Shriners Burns Institute, Boston or Massachusetts General Hospital between 1970 and 1995 was retrospectively analyzed. RESULTS: A total of 16 cases was identified. Among seven subway workers, the mechanism of rail contact was unintentional by a tool, a hand or by falling; no deaths occurred. Among nine non-occupational victims, injuries involved suicide attempts, unintentional falls, or risk-taking behavior. This group suffered greater burn severity, operative procedures, and complications; three deaths occurred. CONCLUSIONS: This is the largest report series of direct electrical trauma from a subway third rail. The high morbidity and mortality from this 600 V contact suggests that the traditional classification of low voltage (< 1000 V) exposure can be subdivided to reflect the serious and lethal potential of intermediate range exposures compared to household range exposures (0-220 V).