The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation.

Cytosolic glutathione transferases (GSTs) were purified from the rat spleen by S-hexyl-GSH-Sepharose chromatography, and two major forms were identified as GSTs 2-2 and 7-7 (GST P). Besides these forms an acidic form (pI 5.8) was purified by chromatofocusing at pH 7-4 and it accounted for about 1% of the total GST activity bound to S-hexyl-GSH-Sepharose. Two-dimensional gel electrophoresis revealed that it is a homodimer (subunit Mr 26,000 with pI 5.8). Immunoblot analysis demonstrated that it was immunologically related to GSTs 2-2 and 1-1, and its N-terminal amino acid was apparently blocked, similarly to other forms of the class Alpha. This form had a low activity towards cumene hydroperoxide or 4-hydroxynon-2-enal, indicating that this form differed from GSTs 10-10 and 8-8 as well as from GSTs 1-1 and 2-2. These results suggest that it is a new form of GST belonging to the class Alpha.     

Immunohistochemically demonstrated increase in glutathione S-transferase species in propylnitrosamine-induced focal proliferative and neoplastic Syrian hamster pancreatic lesions

Immunohistochemical investigation of focal proliferative and neoplastic Syrian hamster pancreatic lesions induced by propylnitrosamine administration revealed a distinct pattern of expression of different molecular forms of glutathione S-transferase (GST) during neoplastic development. Initial increase in levels of GST placental (P) and B forms in early ductal/ductular proliferations and atypical (dysplastic) lesions was followed by a drop in the latter during transition to carcinoma. Unequivocal acinar cells observed within so-called 'pseudoductules' did not share the altered phenotype evident in ductular elements, suggesting their non-involvement in the generation of early lesions. However, the fact that component cells were occasionally of abnormal morphology did not allow the exclusion of acinar cell participation in histogenesis. Elevation of GST-A and C forms was limited to stromal elements surrounding the epithelial lesions and since they were associated with benign, cystic as well as atypical lesions and a similar increase was observed after common duct ligation, they appeared to be non-specific. The results indicated independent control of expression of individual GST forms and suggest that biochemical similarities exist between early, putative preneoplastic lesions induced by propylnitrosamines in the hamster lung, liver (both hepatocellular and intrahepatic bile duct) and pancreas.     

Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.     

Evidence for a multidrug resistance-associated protein 1 (MRP1)-related transport system in cultured rat liver biliary epithelial cells

Cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CF) were studied in rat liver SDVI cells thought to derive from primitive bile ductules, in order to characterize carrier-related membrane transport of organic anions in epithelial cells. Probenecid, a common blocker of anion transport, was found to strongly enhance CF levels in SDVI cells in a dose-dependent manner through inhibition of dye efflux. Such an outwardly-directed transport was demonstrated to be temperature-dependent and down-regulated by various metabolic inhibitors, therefore outlining its requirement for energy; it was shown to be Na+- and membrane potential-independent and inhibited by anionic drugs such as indomethacin, indoprofen and rifamycin B. These functional features are closed to those described for multidrug resistance-associated protein 1 (MRP1) that was furthermore demonstrated, in contrast to P-glycoprotein, to be expressed in SDVI cells and to lower CF accumulation in MRP1-overexpressing drug-resistant tumor cells. These data therefore suggest that active membrane transport of organic anions such as CF occurs in epithelial cells like cultured liver biliary SDVI cells through a MRP1-related efflux system.     

Improved production of recombinant fibroblast growth factor 7 (FGF7/KGF) from bacteria in high magnesium chloride

Because of specificity for both heparin/heparan sulfate and the receptor complex on epithelial cells relative to other fibroblast growth factor (FGF) homologues, there is considerable interest in clinical and commercial applications of FGF7 (also called keratinocyte growth factor or KGF) that require large quantities at reasonable cost. Production of recombinant FGF7 from bacteria suffers from lower yields and recovery relative to FGF1 and FGF2. Fusion of FGF7 at the N-terminus with glutathione-S-transferase (GST) followed by removal of GST by proteolysis while bound to natural ligand heparin improved the intrinsically low yields from Escherichia coli hosts to 3.2 mg per liter per OD(600), which was still only 10% of that for FGF1. Yield of the GST-FGF7 fusion product was improved to about 17 mg per liter per OD(600) in strain BL21(DE3)pLysS by inclusion of 10-100mM magnesium chloride (MgCl(2)) in the culture medium. This improved by about five times the yields of fully active 54ser-FGF7 after proteolytic excision of the GST portion from GST-FGF7 immobilized on heparin-Sepharose. This simple enhancement improves the cost-effectiveness of production of recombinant FGF7 in bacteria for clinical and commercial applications.     

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.     

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.     

Calcium-induced aggregation of neuroendocrine protein 7B2in vitro and its modulation by ATP

To study the behavior of the neuroendocrine polypeptide 7B2 in the presence of calcium, various fragments of this molecule were produced inEscherichia coli as fusion proteins to glutathione S-transferase (GST). Addition of millimolar concentrations of Ca²⁺ to purified preparations of hybrid molecules carrying the N-terminal segment of 7B2 induced precipitation in a manner dependent on protein and cation concentrations. This precipitation occurred at pH 7.5 but not at pH 5.2. It was augmented by 4 and 8 mM ATP, and reduced by 12 and 24 mM ATP. ADP had a similar but weaker effect. Calcium failed to cause precipitation of GST alone or of GST fused to the C-terminal peptide 7B2_156–186. However, when the latter protein was mixed with a GST protein carrying a short fragment of the N-terminal region of 7B2, both proteins were precipitated by calcium. Except for the pH dependence, the behavior of 7B2 fusion proteins in the presence of calcium and adenosine nucleotides are reminiscent of those exhibited by chromogranins and secretogranins, which, like 7B2, are acidic proteins found in the secretory granules of a variety of neuroendocrine cells. As suggested for other granins, this property may underlie the segregation of 7B2 fragments into secretory granules.     

Characterisation of a zeta class glutathione transferase from Arabidopsis thaliana with a putative role in tyrosine catabolism

A glutathione transferase (GST) similar to zeta GSTs in animals and fungi has been cloned from Arabidopsis thaliana using RT-PCR. The Arabidopsis zeta GST (AtGSTZ1) was expressed in Escherichia coli as his-tagged polypeptides, which associated together to form the 50-kDa AtGSTZ1-1 homodimer. Following purification, AtGSTZ1-1 was assayed for a range of activities and compared with other purified recombinant plant GSTs from the phi, tau, and theta classes. AtGSTZ1-1 differed from the other GSTs in showing no glutathione conjugating activity toward xenobiotics and no glutathione peroxidase activity toward organic hydroperoxides. Uniquely among the plant GSTs, AtGSTZ1-1 showed activity as a maleylacetone isomerase (MAI). This glutathione-dependent reaction is analogous to the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate, which occurs in the course of tyrosine catabolism to acetoacetate and fumarate. Thus, rather than functioning as a conventional GST, AtGSTZ1-1 appears to be involved in tyrosine degradation. In addition to the MAI activity, the AtGSTZ1-1 also catalyzed the glutathione-dependent dehalogenation of dichloroacetic acid to glyoxylic acid. This latter activity was used to demonstrate the presence of functional AtGSTZ1-1 inplanta.     

Anion recognition by thiostrepton

A bicyclic polypeptide antibiotic thiostrepton forms both 1:1 and 1:2 complexes with anions (as tetrabutylammonium salts) in organic solvents with K2 >K1 for F- and K2Cl-, Br-, HSO4-, H2PO4-, but in CHCl3 they follow a different order: Cl- approximatelyHSO4- >F- approximately AcO- > Br > H2PO4-. The binding mode of anions to thiostrepton is discussed on the basis of solvent effects on the complexation selectivity.     

Human Recombinant B7-H3 Expressed in E.colt Enhances T Lymphocyte Proliferation and IL-10 Secretion in Vitro

Since it was proposed in 1970, the two-signal hypothesis for T lymphocyte activation became widely accepted, which elucidated T cells required two distinct signals for optimal T helper precursor cell expansion. This model hypothesized that peptides presen…     

Human recombinant B7-H3 expressed in E. coli enhances T lymphocyte proliferation and IL-10 secretion in vitro

To explore the biofunctions of human B7-H3 on activated T lymphocyte, the gene of human B7-H3 encoding the extracellular region (IgV-like and IgC-like domains) was obtained by RT-PCR from human lung cells and subcloned into the prokaryotic expression vector pGEX-5X-3 to express glutathione S-transferase (GST) fusion protein. A 49 kD fusion protein (named as GST/hB7-H3 hereafter) was induced by IPTG and purified by standard methods reported in prokaryotic system. In the presence of the first signal imitated by anti-CD3 monoclonal antibody, T lymphocyte proliferation was observed by incubating purified T cells with soluble GST/hB7-H3 fusion protein by MTT assay. The concentrations of IFN-γ and IL-10 in the supernatants of T cells were determined by ELISA. The results showed that the GST/hB7-H3 protein produced in bacteria had modest biological activities to proliferate the T lymphocyte and enhance IFN-γ as well as IL-10 secretion.     

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines

Enzyme-linked immunoassays (ELISAs) based on the double-antibody sandwich technique have been developed for the quantitative analysis of the major human cytosolic class Pi, Mu and Alpha glutathione transferases (GSTs). The procedures were optimized with respect to antibody concentration for coating of plates as well as other parameters in order to achieve high sensitivity and accuracy. No cross-reactivity was detected between members of the three different classes of GSTs or among the Mu class GSTs M2-2, M3-3 and M4-4 with the ELISA for GST M1-1. The ELISAs have been applied to establish the cytosolic GST profiles of 10 cell lines and to monitor the plasma GST levels in cancer patients. The results revealed that the class Pi GST was the dominant isoenzyme in six (LS 174T, HCT-8, Hu 549 Pat, K-562, U-937 and Hu 549) out of nine tumor cell lines and immortalized hepatocytes (Chang Liver). The isoenzymes A1-1 and M1-1 were determined to be the major GST components in Hep G2 and HeLa cells, respectively. In a clinical study, the majority of the patients with urinary bladder cancer were found to have increased plasma levels of both GST A1-1 and GST P1-1 (10/15), while patients with renal cancer frequently showed increases only in GST P1-1 (5/8). The results demonstrate that the ELISAs are suitable for analyzing GST phenotypes in both normal and tumor cells and in monitoring plasma levels of GSTs in cancer patients.     

Refolding of the Escherichia coli expressed extracellular domain of alpha 7 nicotinic acetylcholine receptor.

Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand-binding sites. The ECD of the alpha 7 subunit of the homo-oligomeric alpha 7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat alpha 7 ECDs (amino-acid residues approximately 1-210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein [Fischer, M., Corringer, P., Schott, K., Bacher, A. & Changeux, J. (2001) Proc. Natl Acad. Sci. USA 98, 3567-3570] and glutathione S-transferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E., Chiodini, F., Bertrand, D., Methfessel, C. & Tsetlin, V. (2001) J. Biol. Chem. 276, 15810-15815]. However, these proteins exist in solution mostly as high-molecular mass aggregates rather than monomers or oligomers. In the present work it is found that refolding of GST-alpha 7-(1-208) protein in the presence of 0.1% SDS considerably decreases the formation of high-molecular mass aggregates. The C116S mutation in the alpha 7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions. This mutation slightly increased the affinity of the protein for alpha-bungarotoxin (from Kd approximately 300 to 150 nm). Gel-permeation HPLC was used to isolate the monomeric form of the GST-alpha 7-(1-208) protein and its mutant almost devoid of SDS. CD spectra revealed that the C116S mutation considerably increased the content of beta structure and made it more stable under different conditions. The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the alpha 7 ECD in an oligomeric form.     

Identification of immunodominant Th1-type T cell epitopes from Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candidate

Th1-type cytokines produced by the stimulation of Th 1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection. Schistosoma mansoni 28 kDa glutathione-S-transferase （Sm28GST）, a major detoxification enzyme, has been recognized as a vaccine candidate and a phase II clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST （Sj28GST） have shown immune protection against the parasite infection. In the present study, six candidate peptides （P1, P2, P3, P4, P7 and P8） from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope （190-211 aa） identified from Sm28GST was selected and named P9. The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c（＋）/BL21（DE3） system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6 （73-86 aa） generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ, and IL-2. Therefore, P6 is a functional Thl-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Thl-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.     

The P2X7 receptor: Shifting from a low- to a high-conductance channel — An enigmatic phenomenon?

The general structure of the P2X7 receptor (P2X7R) is similar to the structure of other P2X receptor family members, with the exception of its C terminus, which is the longest of this family. The P2X7R activates several intracellular signaling cascades, such as the calmodulin, mitogen-activated protein kinase and phospholipase D pathways. At low concentrations of ATP (micromolar range), P2X7R activation opens a cationic channel, similarly to other P2X receptors. However, in the presence of high concentrations of ATP (millimolar range), it opens a pathway that allows the passage of larger organic cations and anions. Here, we discuss both the structural characteristics of P2X7R related to its remarkable functions and the proposed mechanisms, including the dilation of the endogenous pore and the integration of another channel. In addition, we highlight the importance of P2X7R as a therapeutic target.     

Specific inactivation of glutathione S-transferases in class Pi by SH-modifiers

Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs.