Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin

In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon 1 h incubation with 100 microM quercetin or 2 h incubation with 25 microM quercetin, whereas 1 and 10 microM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LC-MS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LC-MS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47.     

Aphid saliva

Within the Aphidoidea, most species of Aphididae, as long as they are in small numbers and not carrying plant viruses, do little perceptible damage to their food plants. In species that cause toxicoses, it is usually assumed that some component of the saliva must be responsible. Paradoxically, however, the salivary enzymes of Aphididae are similar to those that already occur in plants – oxidases and enzymes that depolymerize polysaccharides – and the salivary enzymes are injected in very small amounts relative to their counterparts in the plant. Damage to plants triggers defensive, biochemical responses, and it is suggested that the injected enzymes serve mainly to divert or counter responses at the immediate interface of stylets and plant tissues. The saliva of Aphididae contains non-enzymic, reducing compounds which, in the presence of oxidases, can combine with and inactivate defensive phytochemicals – including those released in response to damage and transported in the phloem sieve tube sap on which Aphididae feed. Salivary and gut oxidases deactivate ingested phytochemicals by oxidative polymerization. Aphididae inject saliva into sieve tubes before sustained ingestion of sap, and this saliva has been presumed to condition the sieve tubes, but in what way remains unclear. It is suggested that there is a dynamic biochemical interaction between aphids and plants; that the interaction is usually well balanced for most of the Aphididae; hence, no outcome is readily observable. Where a significant imbalance occurs, however, either the aphid is unable to feed, i.e. the plant is resistant, and/or the aphid does not effectively counter a hypersensitive response. Not all plant responses are disadvantageous to aphids. Gall-forming Aphidoidea trigger and control abnormal growth in the plant to the insects' advantage, possibly by eliciting vigorous oxidation in selective meristematic tissues, thereby limiting supply of molecular oxygen and inhibiting oxygen-dependent growth-controls. Current problems and possible approaches for further research are reviewed.     

Salivary hormones: a new aspect of oral physiology

Aside from the digestive enzymes the submandibular salivary glands (SSG) synthetize other polypeptides, detected also in saliva, with varied biological activity; NGF and EGF are the knowest. However, over the last decade, steroids hormones have been also found out in the saliva at the same concentrations that the free plasma fraction. The origin of these hormones is largely discussed and certain authors have even proposed a local synthesis for them. This matter, is of clinical interest because gingiva and buccal tissues are knowingly sensitive to steroids. Besides, woman ovulation appears to be monitored through progesterone fluctuations in saliva. Another kind of salivary substances is formed by the neuropeptides of the gut-brain axis, mainly VIP and SRIF. The former likely of nervous origin seems to be involved in the atropine-resistant salivary secretion, whereas the latter-likely of SSG origin--appears as a factor associated with glycemia control.     

Modulating effects of prostaglandins on parasympathetic-mediated secretory activities of rat salivary glands

Exogenously administered PGE₁ or PGE₂, like atropine, markedly decreased both the flow and calcium concentration of parasympathetically evoked rat parotid saliva: PGF_2α was less effective. Despite the fact that prostaglandins greatly reduced the Ca concentration of nerve-evoked saliva, they did not change the glandular Ca concentration of either control or parasympathetically stimulated parotid glands. Prostaglandins (20 μg/kg, i.a.) decreased the Na or K concentration of nerve-evoked parotid saliva, but at lower doses had no significant effect. PGE₁, PGE₂, PGF_2α or atropine markedly decreased flow rates of similarly evoked rat submandibular saliva. Prostaglandins and atropine, however, decreased the Na concentration and increased the K concentration of parasympathetically evoked submandibular saliva. PGF_2α, like atropine, increased the Ca concentration of such saliva. Drug vehicle, ethanol, slightly decreased the flow of both parotid and submandibular saliva but not the ion secretion. Endogenous prostaglandins themselves may not play a role in a secretory activities during parasympathetic nerve stimulation of rat salivary glands, since administration of indomethacin, an inhibitor of prostaglandins biosynthesis, prior to or during nerve stimulation did not significantly alter nerve-evoked salivary secretion. The mechanisms by which prostaglandins modulate secretory responses of salivary glands during parasympathetic stimulation are not understood.     

Modulation of glutathione transferase P1-1 activity by retinoic acid in neuroblastoma cells.

The ability of retinoic acid to modulate glutathione S-transferase P1-1 (GSTP1-1) activity has important implications both for cancer prevention and for anticancer therapy. We investigated GSTP1-1 expression and activity in the human neuroblastoma cell line SK-N-BE(2) (genotype A*/B*) under basal conditions and during 48-h incubation with 0.1 microM all-trans-retinoic acid. The steady-state levels of glutathione transferase P1-1 mRNA and protein during 48-h incubation with all-trans-retinoic acid did not increase substantially, but we detected a significant reduction of GSTP1-1 specific activity. This reduction in enzymatic activity could not be ascribed to a differential action of retinoic acid on the gene variants A* and B*; indeed, the two GSTP1-1 isoforms have different affinities toward 1-chloro-2,4-dinitrobenzene (CDNB), while we found a substantial invariance of the K(m) (CDNB) in the cytosol during retinoid treatment. A modulatory effect of retinoic acid on other enzymes involved in glutathione transferase P1-1 metabolism, such as the retinoic acid-induced tissue trans-glutaminase, might be hypothesized, as well as a direct inactivation of GSTP1-1 by the oxidative stress that characterizes the early phases of apoptosis.     

α1-Antitrypsin and α2-macroglobulin do not inhibit the kinin-releasing activity of kallikreins from human urine and saliva

α₁-Antitrypsin, α₂-macroglobulin and low-molecular weight kininogen were isolated from human serum and kallikreins from human urine and saliva.α₁-Antitrypsin and α₂-macroglobulin inhibited the activity of trypsin in releasing kinin from low-molecular weight kininogen, due to their binding with the enzyme, but did non inhibit or bind with urinary and salivary kallikreins.     

Suppression of Radiation-Induced Salivary Gland Dysfunction by IGF-1

Background

Radiation is a primary or secondary therapeutic modality for treatment of head and neck cancer. A common side effect of irradiation to the neck and neck region is xerostomia caused by salivary gland dysfunction. Approximately 40,000 new cases of xerostomia result from radiation treatment in the United States each year. The ensuing salivary gland hypofunction results in significant morbidity and diminishes the effectiveness of anti-cancer therapies as well as the quality of life for these patients. Previous studies in a rat model have shown no correlation between induction of apoptosis in the salivary gland and either the immediate or chronic decrease in salivary function following γ-radiation treatment.

Methodology/Principal Finding

A significant level of apoptosis can be detected in the salivary glands of FVB mice following γ-radiation treatment of the head and neck and this apoptosis is suppressed in transgenic mice expressing an activated mutant of Akt (myr-Akt1). Importantly, this suppression of apoptosis in myr-Akt1 mice preserves salivary function, as measured by saliva output, three and thirty days after γ-radiation treatment. In order to translate these studies into a preclinal model we found that intravenous injection of IGF1 stimulated activation of endogenous Akt in the salivary glands in vivo. A single injection of IGF1 prior to exposure to γ-radiation diminishes salivary acinar cell apoptosis and completely preserves salivary gland function three and thirty days following irradiation.

Conclusions/Significance

These studies suggest that apoptosis of salivary acinar cells underlies salivary gland hypofunction occurring secondary to radiation of the head and neck region. Targeted delivery of IGF1 to the salivary gland of patients receiving head and neck irradiation may be useful in reducing or eliminating xerostomia and restoring quality of life to these patients.     

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

This paper reports that the acetylation of lysine ε-NH₃⁺ groups of α-amylase—one of the most important hydrolytic enzymes used in industry—produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild-type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90°C in solutions containing 100-mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated α-amylase toward inactivation is attributed to the increased net negative charge of α-amylase that resulted from the acetylation of lysine ammonium groups (lysine ε-NH₃⁺ → ε-NHCOCH₃). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial α-amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.     

Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel

Large-conductance voltage- and Ca²⁺-gated K⁺ channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0–αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2–β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2–β4; and the disulfide pattern is 1–8, 3–4, and 5–6. Presumably, Cys 2 cross-links to Cys 7 in all other β2–β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0–S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.     

Taking the Starch out of Oral Biofilm Formation: Molecular Basis and Functional Significance of Salivary α-Amylase Binding to Oral Streptococci

α-Amylase-binding streptococci (ABS) are a heterogeneous group of commensal oral bacterial species that comprise a significant proportion of dental plaque microfloras. Salivary α-amylase, one of the most abundant proteins in human saliva, binds to the surface of these bacteria via specific surface-exposed α-amylase-binding proteins. The functional significance of α-amylase-binding proteins in oral colonization by streptococci is important for understanding how salivary components influence oral biofilm formation by these important dental plaque species. This review summarizes the results of an extensive series of studies that have sought to define the molecular basis for α-amylase binding to the surface of the bacterium as well as the biological significance of this phenomenon in dental plaque biofilm formation.     

The secretion and action of the digestive enzymes of the salivary glands of the blowfly, Calliphora

The salivary glands of adult Calliphora contain enzymes which hydrolyze starch, sucrose and trehalose. Amylase and sucrase are shown to be secretory enzymes, while trehalase remains in the gland. Results of electrophoretic and ultrastructural studies suggest that protein secretion is confined to the abdominal region of the gland. Secretion of both fluid and protein occurs from a single type of cell. While a fly is feeding on solid sugar, amylase and sucrase are lost from the gland and appear in saliva, while the level of trehalase in the gland increases slightly. The mixture of food and saliva passes mainly to the crop where carbohydrate is digested by the salivary enzymes.     

The role of GSTM1, GSTT1, GSTP1, and OGG1 polymorphisms in type 2 diabetes mellitus risk: a case-control study in a Turkish population

The aim of the present study was to investigate the role of some polymorphisms in GSTs (GSTM1, GSTT1 and GSTP1) which are very important protective mechanisms against oxidative stress and in OGG1 gene which is important in DNA repair, against the risk of type 2 diabetes mellitus (T2DM). 127 T2DM and 127 control subjects were included in the study. DNA was extracted from whole blood. Analyses of GSTM1 and GSTT1 gene polymorphisms were performed by allele specific PCR and those of GSTP1 Ile105Val and OGG1 Ser326Cys by PCR-RFLP. Our data showed that GSTM1 null genotype frequency had a 2-6 times statistically significant increase in a patient group (OR=3.841, 95% CI=2.280-6.469, p<0.001) but no significance with GSTT1 null/positive and GSTP1 Ile105Val genotypes was observed. When T2DM patients with OGG1 Ser326Cys polymorphism were compared with patients with a wild genotype, a 2-3 times statistically significant increase has been observed (OR 1.858, 95% CI=1.099-3.141, p=0.021). The combined effect of GSTM1 null and OGG1 variant genotype frequencies has shown to be statistically significant. Similarly, the risk of T2DM was statistically increased with GSTM1 null (OR=3.841, 95% CI=2.28-6.469), GSTT1 null+GSTP1 (H+M) (OR=4.118, 95% CI=1.327-12.778) and GSTM1 null+OGG1 (H+M) (OR=3.322, 95% CI=1.898-5.816) and GSTT1 null+OGG1 (H+M) (OR=2.179, 95% CI=1.083-4.386) as compared to the control group. According to our study results, it has been observed that the combined evaluation of GSTM1-GSTT1-GSTP1 and OGG1 Ser326Cys gene polymorphisms can be used as candidate genes in the etiology of T2DM, especially in the development of T2DM.     

The cynomolgus monkey (Macaca fascicularis) is the best animal model for the study of steroid glucuronidation

Intense research efforts performed during the past decade clearly established the major role of glucuronidation and uridine-diphospho-glucuronosyltransferase (UGT) enzymes for steroid metabolism in humans. However, a clear understanding of the physiological importance of this metabolic process requires in vivo studies. Numerous evidences ascertain that simians are the most appropriate animal models for such studies. Indeed human and monkey have a similar pattern of steroidogenesis, unlike common laboratory mammals such as rat or mouse. Furthermore, human and monkey are unique in having high levels of circulating androsterone glucuronide and androstane-3α-diol glucuronide (3α-Diol-G). In addition, characterization of eight monkey UGT proteins demonstrated the similarity of their conjugation activity toward steroid hormones. Like human ones, monkey enzymes are expressed in steroid target tissues, where they preferentially glucuronidate androgen and estrogen metabolites. In monkey tissues, immunohistochemical studies demonstrated that UGT2B proteins are expressed in a cell-type specific manner in ovary and kidney, where they control androgens and aldosterone inactivation. These results identify the cynomolgus monkey as an appropriate animal model for the determination of cellular localization of UGT enzymes in steroid target tissues and for the identification of endogenous or exogenous stimuli affecting steroid glucuronidation.     

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion

Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1β (IL-1β) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.     

Proteases and nucleases involved in the biphasic digestion process of the brown marmorated stink bug,Halyomorpha halys (Hemiptera: Pentatomidae)

Management of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), an invasive, agricultural pest in the United States, has presented significant challenges. This polyphagous insect uses both extra‐oral and gut‐based digestion thwarting protein‐ or nucleotide‐based control strategies. The objective of this study was to biochemically characterize the digestive enzymes (proteases and nucleases) from the saliva, salivary gland and the gut of H. halys. Enzyme profiles for the two tissues and saliva radically differ: The pH optimum for proteases in the gut was six, with cysteine proteases predominant. In contrast, the alkaline pH optima for protease activity in the salivary gland (8–10) and saliva (7) reflected abundant serine protease and cathepsin activities. RNase enzymes were most abundant in saliva, while dsRNase and DNase activities were higher in the salivary gland and saliva compared to those in the gut. These very different enzyme profiles highlight the biphasic digestive system used by this invasive species for efficient processing of plant nutrients. Knowledge of H. halys digestive physiology will allow for counteractive measures targeting digestive enzymes or for appropriate protection of protein‐ or nucleotide‐based management options targeting this pest.     

Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation

The dual-functioning antioxidant enzyme peroxiredoxin VI (Prdx6) detoxifies lipid peroxides particularly in biological membranes, and its peroxidase function is activated by glutathione S-transferase Pi (GSTP). The GSTP gene is polymorphic in humans, with the wild-type GSTP1-1 A (Ile105, Ala114) and three variants: GSTP1-1B (Ile105Val, Ala114), GSTP1-1C (Ile105Val, Ala114Val), and GSTP1-1D (Ile105, Ala114Val). The focus of this study was to determine the influence of these polymorphisms on Prdx6 peroxidase function. Using extracellular generation of OH radicals and fluorescence (DPPP dye) detection, we found a fast (∼300 s) onset of lipid peroxidation in membranes of MCF-7 cells transfected with a catalytically inactive Y7F mutant of GSTP1-1 and either GSTP1-1B or GSTP1-1D. However, this effect was not detected in cells expressing either GSTP1-1A or GSTP1-1C. Imaging of DPPP-labeled MCF-7 cells showed fluorescence localized in the plasma membrane, but intensity was substantially diminished in the GSTP1-1 A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1 A-, GSTP1-1B-, and GST1-1D-expressing cells OH generation resulted (after 36 h) in plasma membrane-permeability-related cell death, whereas GSTP1-1A- and GSTP1-1C-expressing cells had significantly better survival. We used FRET analyses to measure in vitro binding of purified GSTP1-1 allelic variant proteins to purified recombinant Prdx6. The affinities for Prdx6 binding to GSH-loaded GSTP1-1's either mirrored their observed peroxidase activities (using phospholipid hydroperoxide as a substrate), GSTP1-1A>GSTP1-1C (K_D=51.0 vs 57.0 nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (K_D=101.0 (94.0) nM). In silico modeling of the GSTP1-1–Prdx6 heterodimer revealed that the sites of GSTP1-1 polymorphism (Ile105 and Ala114) are in close proximity to the binding interface. Thus, there is a hierarchy of effectiveness for polymorphic variants of GSTP1-1 to regulate Prdx6 peroxidase function, a feature that may influence human population susceptibilities to oxidant stress.     

Glutathione S-transferase M1, T1 and P1 genetic polymorphisms, cigarette smoking and gastric cancer risk

Glutathione S-transferases (GSTs) belong to a superfamily of detoxification enzymes that provide critical defences against a large variety of chemical carcinogens and environmental toxicants. GSTs are present in most epithelial tissues of the human gastrointestinal tract. We investigated associations between genetic variability in specific GST genes (GSTM1, GSTT1 and GSTP1), the interaction with cigarette smoking and susceptibility to gastric cancer. The GSTM1, GSTT1 and GSTP1 polymorphisms were determined using real-time polymerase chain reaction (PCR) and fluorescence resonance energy transfer with Light Cycler Instrument. The study included 70 patients with gastric cancer and 204 controls. Associations between specific genotypes and the development of gastric cancer were examined by use of logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI). The GSTM1 homozygous null genotype was associated with an increased risk of developing gastric cancer (OR = 1.73; 95% CI = 1.10-3.04). GSTT1 homozygous null genotype and GSTP1 genotypes were not associated with the risk of gastric cancer. Also there was no difference between cases and controls in the frequency of val-105 and ile-105 alleles (p = 0.07). After grouping according to smoking status, GSTM1 null genotype was associated with an increased gastric cancer risk for smokers (OR = 2.15; 95% CI, 1.02-4.52). There were no significant differences in the distributions of any of the other GST gene combinations. Our findings suggest that the GSTM1 null genotype may be associated with an increased susceptibility to gastric cancer.     

Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin

The inactivation mechanism(s) of human glutathione S-transferase P1-1 (hGST P1-1) by the catechol metabolite of Premarin estrogens, 4-hydroxyequilenin (4-OHEN), was (were) studied by means of site-directed mutagenesis, electrospray ionization mass spectrometric analysis, titration of free thiol groups, kinetic studies of irreversible inhibition, and analysis of band patterns on nonreducing sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The four cysteines (Cys 14, Cys 47, Cys 101, and Cys 169 in the primary sequence) in hGST P1-1 are susceptible to electrophilic attack and/or oxidative damage leading to loss of enzymatic activity. To investigate the role of cysteine residues in the 4-OHEN-mediated inactivation of this enzyme, one or a combination of cysteine residues was replaced by alanine residues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169A mutants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate 1-chloro-2,4-dinitrobenzene (CDNB). However, the Cys 47 mutation did not significantly affect the rate of catalysis since V(max) values of the mutants were similar or higher compared to that of wild type. Electrospray ionization mass spectrometric analyses of wild-type and mutant enzymes treated with 4-OHEN showed that a single molecule of 4-OHEN-o-quinone attached to the proteins, with the exception of the C14A/C47A/C101A mutant where no covalent adduct was detected. 4-OHEN also caused oxidative damage as demonstrated by the appearance of disulfide-bonded species on nonreducing SDS--PAGE and protection of 4-OHEN-mediated enzyme inhibition by free radical scavengers. The studies of thiol group titration and irreversible kinetic experiments indicated that the different cysteines have distinct reactivity for 4-OHEN; Cys 47 was the most reactive thiol group whereas Cys 169 was resistant to modification. These results demonstrate that hGST P1-1 is inactivated by 4-OHEN through two possible mechanisms: (1) covalent modification of cysteine residues and (2) oxidative damage leading to proteins inactivated by disulfide bond formation.     

Evaluation of the Galα1-3Gal Epitope as a Host Modification Factor Eliciting Natural Humoral Immunity to Enveloped Viruses

Human sera contain high levels of natural antibody (Ab) to Galα1-3Gal, a terminal glycosidic structure expressed on the surface of cells of mammals other than Old World primates. Incorporation of this determinant onto retroviral membranes by passage of viruses in cells encoding α-1-3-galactosyltransferase (GT) renders retroviruses sensitive to lysis by natural Ab and complement in normal human serum (NHS). Plasma membrane-budding viruses representing four additional virus groups were examined for their sensitivities to serum inactivation after passage through human cell lines that lack a functional GT or human cells expressing recombinant porcine GT. The inactivation of lymphocytic choriomeningitis virus (LCMV) by NHS directly correlated with host modification of the virus via expression of Galα1-3Gal and was blocked by incorporation of soluble Galα1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galα1-3Gal (anti-Gal Ab) were tested for resistance to LCMV passaged in GT-expressing cells. Resistance was not observed, but in vitro analyses of the mouse immune sera revealed that the antiviral activity of the sera was insufficient to eliminate LCMV infectivity on its natural targets of infection, macrophages, which express receptors for Ab and complement. Newcastle disease virus and vesicular stomatitis virus (VSV) were inactivated by NHS regardless of cell passage history, whereas Sindbis virus (SV) passaged in human cells resisted inactivation. Both VSV and SV passaged in Galα1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galα1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galα1-3Gal differ in their sensitivities to NHS and that a potent complement source, such as that in NHS, is required for efficient inactivation of sensitive viruses in vitro and in vivo.