The physiological indices of pre-examination stress

Overexpression of Bcl-2 and related anti-apoptotic gene products has been shown to increase the intracellular concentration of the antioxidant tripeptide glutathione in neuronal and hematopoietic cells. A similar examination of HeLa cells that stably overexpress Bcl-2 (Bcl-2/HeLa) demonstrated that the reduced form of glutathione (GSH) was increased by 60% compared to control cells (80 nmol GSH/mg protein compared to 50 nmol GSH/mg). Expression of gamma-glutamylcysteine synthetase, the rate limiting enzyme for glutathione synthesis was found to be independent of Bcl-2 overexpression, as determined by Northern blot analysis and immunoprecipitation of [35-S]-labeled enzyme. Bcl-2 overexpression did not alter the rate of GSH biosynthesis, measured under steady state conditions. Thus, the increase in GSH concentration was not the result of increased synthesis. Two activities have been described which govern efflux of reduced glutathione (GSH), RsGshT known as the sinusoidal transporter and RcGshT, known as the canalicular transporter. Both are low affinity, bidirectional, ATP and Na-independent. Consistent with expression of sinusoidal activity, DTT was found to stimulate GSH efflux while the amino acid methionine inhibited efflux in both HeLa and Bcl-2/HeLa cells. However, methionine-dependent inhibition of efflux was found to be significantly increased by expression of Bcl-2. To test the prediction that the increase in GSH observed in Bcl-2/HeLa cells was mediated by methionine; Bcl-2/HeLa cells were cultured for 24 hrs in methionine-free growth medium. Under these conditions, the GSH concentration of the Bcl-2/HeLa cells dropped to the level observed in HeLa cells (50 nmol GSH/mg protein). These studies suggest that overexpression of Bcl-2 increases GSH levels by altering methionine-dependent GSH efflux, an activity associated in HeLa cells with expression of the RsGshT transporter.     

A single mouse glutathione synthetase gene encodes six mRNAs with different 5' ends

To understand more about the role of glutathione (GSH) in metabolism, we have cloned both cDNA and genomic sequences for mouse glutathione synthetase (GSH syn), the enzyme that catalyzes the last step in the synthesis of glutathione. The mouse cDNA contains an open reading frame (ORF) of 474 aa and shares 64 and 95% deduced amino acid sequence identity with Xenopus cDNA and rat cDNA, respectively. The cDNA complements Schizosaccaromyces pombe strains deficient in GSH syn. The gene is a single-copy gene spanning approximately 30 kb and is composed of at least 15 exons. Steady-state RNA levels and enzyme activity levels are highest in kidney, about 3-fold lower in liver, and 8- to 10-fold lower in lung and brain. We have identified six different GSH syn RNAs: three, termed types A1, A2, and A3, have different 5' ends that localize to different sites in the gene, but appear to encode the same protein (474 aa). Types B, C1, and C2 all have unique 5' ends and type-specific ORFs, which are shorter than that for types A1, A2, and A3. In liver only type A1 GSH syn RNA is detectable, while in kidney 90% of GSH syn RNA is type A1 and types B and C account for about 10%.     

Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M(r) = 72,550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K(m) for sulphate was 6.9 microM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.     

Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA

Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.     

Thiol-disulfide effects on hepatic glutathione transport. Studies in cultured rat hepatocytes and perfused livers

In cultured rat hepatocytes, cystine led to an inhibition of GSH efflux by lowering the Vmax by approximately 35% without affecting the Km. The cystine-mediated inhibition of GSH efflux was rapid in onset (< 1 h), with near maximum effect at 0.1 mM. Inhibition was still observed when cystine uptake was prevented. Cystine and sulfobromophthalein-GSH, a selective inhibitor of sinusoidal transport of GSH, did not exhibit additive inhibitory effects on GSH efflux. Depletion of ATP or membrane depolarization after cystine treatment were excluded as potential mechanisms. DTT not only reversed the cystine-mediated inhibition of GSH efflux, it stimulated GSH efflux up to 400-500%. The DTT effect was immediate in onset, reaching maximum after 30 min, and was partially reversed by cystine, suggesting that the two share a common site(s) of action. DTT treatment did not alter cellular ATP levels or change the membrane potential. In cultured hepatocytes, DTT treatment increased the Vmax of GSH efflux by approximately 500% without affecting the Km. Inhibition of microtubular function and vesicular acidification did not affect basal or DTT stimulated efflux. Both cystine and DTT effects on sinusoidal GSH efflux were confirmed in perfused livers. In summary, the capacity of the sinusoidal GSH transporter is markedly influenced by thiol-disulfide status.     

Molecular regulation of sinusoidal liver bile acid transporters during cholestasis

Impairment of the hepatic transport of bile acids and other organic anions will result in the clinically important syndrome of cholestasis. Cloning of a number of specific hepatic organic anion transporters has enabled studies of their molecular regulation during cholestasis. The best characterized transport system is a 50-51 kDa sodium-dependent taurocholate cotransporting polypeptide (ntcp), which mediates the sodium-dependent uptake of conjugated bile acids at the sinusoidal plasma membrane of hepatocytes. Under physiologic conditions and after depletion of biliary constituents, ntcp remains constitutively expressed throughout the liver acinus. However, both function and expression of ntcp are rapidly down-regulated in rat liver in various models of experimental cholestasis, such as cholestasis induced by common bile duct ligation, estrogen, endotoxin or cytokine treatment. In addition to ntcp, the sinusoidal organic anion transporting polypeptide oatp-1 is also down-regulated at the protein and steady-state mRNA levels in estrogen-cholestasis, but does not affect sodium-independent uptake of taurocholate. The regulation of a recently cloned member of the organic anion transporter family (oatp-2), which is highly expressed in liver, remains to be studied under cholestatic conditions.     

A novel glutathione peroxidase in bovine eye. Sequence analysis, mRNA level, and translation

Bovine ciliary body contains a selenium-independent glutathione peroxidase (GPX) with a molecular mass of about 100 kDa that is composed of four identical subunits and exhibits no glutathione S-transferase activity. In this study, we isolated cDNA clones and determined the nucleotide sequence to deduce the primary structure of the enzyme. The cDNA contained 672 base pairs encoding a polypeptide with an estimated molecular mass of 25,064 Da. Translation of bovine ciliary mRNA produced a protein which was immunologically indistinguishable from GPX and showed high enzyme activity. The encoded amino acid sequence of the protein was 95% identical with that of a human keratinocyte gene product expressed in response to keratinocyte growth factor. It also showed sequence identity to bacterial alkyl hydroperoxide reductases and thiol specific antioxidant enzymes. GPX mRNA level was highest in the ciliary body, followed by the retina and iris. In various rat organs, the level of GPX mRNA was highest in the lung, followed by the muscle, liver, eye, heart, testis, thymus, kidney, and spleen. A very low level of mRNA was detected in the brain. Enzyme-linked immunosorbent assay with an antibody raised against the NH2-terminal sequence of GPX detected GPX protein in all rat tissues examined.     

Refined structures of bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment

The structure and expression pattern of a human gene located within a homozygously deleted region of a metastatic prostate cancer have been characterized. Multiple cDNA fragments of this gene were isolated by hybrid capture with yeast artificial chromosome clones covering the deletion region. Eleven coding exons spanned 205-220 kb of the 730- to 970-kb deletion. The predicted amino acid sequence was 43% identical to that of an anonymous Caenorhabditis elegans gene and 20% identical to an accessory or regulatory subunit of the oligosaccharyltransferase enzyme complex in Saccharomyces cerevisiae. Hydrophobicity profiles of all three gene products were similar and showed four putative membrane-spanning domains in the molecules' C-terminal halves, suggesting a general conservation of function. The gene was expressed as an approximately 1.5-kb mRNA in most nonlymphoid human cells/tissues including prostate, lung, liver, and colon. Expression was detected in many epithelial tumor cell lines, but was undetectable by Northern blot or RT-PCR in 14 of 15 colorectal, 1 of 8 lung, and 1 of 4 liver cancer cell lines. Lack of expression in tumor cell lines was highly correlated with hypermethylation of a CpG island located at the gene's 5' end. These findings form a basis for further work on this candidate tumor suppressor gene.     

Glutathione-dependent factors and inhibition of rat liver microsomal lipid peroxidation

The effects of reduced glutathione (GSH) and glutathione disulfide (GSSG) on lipid peroxidation were investigated in rat liver microsomes containing deficient or adequate amounts of alpha-tocopherol (alpha-TH). Rates of formation of thiobarbituric acid reactive substances (TBARS) as well as rates of consumption of alpha-TH and O2 were decreased by GSH and were more pronounced in the NADPH-dependent assay system than in the ascorbate-dependent system. The GSH-dependent inhibition of lipid peroxidation was potentiated by GSSG in the NADPH-dependent assay system, but it had no effect in the nonenzymatic system. Diphenyliodonium chloride, an inhibitor of NADPH cytochrome P-450 reductase, completely prevented lipid peroxidation in the NADPH-dependent assay system whereas it had no effect on the ascorbate-dependent system. This is further evidenced by the fact that purified rat liver microsomal NADPH cytochrome P-450 reductase (EC 1.6.2.4) was inhibited approximately 24% and 52% by 5 mM GSH and 5 mM GSH + 2.5 mM GSSG, respectively. Glutathione disulfide alone had no effect on reductase activity. Similarly, other disulfides such as cystine, cystamine and lipoic acid were without effect on reductase activity. These results clearly delineate different mechanisms underlying the combined effects of GSH and GSSG on microsomal lipid peroxidation in rat liver. One mechanism involves recycling of microsomal alpha-TH by GSH during oxidative stress via a labile protein, ostensibly associated with "free radical reductase" activity. A second glutathione-dependent mechanism appears to be mediated through the inhibition of NADPH cytochrome P-450 reductase. The enhanced inhibition by GSH + GSSG of microsomal lipid peroxidation in the NADPH-dependent assay system suggests suppression of the initiation phase at the level of NADPH cytochrome P-450 reductase which is independent of microsomal alpha-TH.     

Increase of the background human exposure to mercury through fish consumption due to gold mining at the Tapajós River region, Pará State, Amazon

Plasma and lipoprotein lipid composition and endogenous hepatic antioxidant status were investigated in hypertensive, 14-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats fed a standard commercial rat chow. Total plasma calcium and magnesium concentrations were similar between both rat strains; however, systolic blood pressure in SHR was greater than in WKY at 13 weeks of age (197 +/- 12 vs. 132 +/- 14 mmHg; p < or = 0.05), confirming hypertension in SHR. Total plasma cholesterol and triacylglycerol concentrations were lower (p < or = 0.05) in SHR compared with WKY. A lower (p < 0.05) HDL cholesterol level in SHR plasma resulted in a higher LDL to HDL cholesterol ratio compared with WKY counterparts. No significant differences in the relative proportion of HDL apolipoprotein A-I fraction were observed between SHR and WKY. Both SHR VLDL and HDL triacylglycerol fractions were lower (p < 0.05) in SHR than WKY. Analysis of liver antioxidant enzyme activities showed no differences in rat liver superoxide dismutase (SOD), but lower (p < 0.05) liver glutathione peroxidase (GSH-Px) activity in SHR. However, liver glutathione (GSH) levels were similar in SHR and WKY counterparts. A possible compensatory effect to the oxidative status of SHR was suggested by the significant (p < 0.05) increase in both liver catalase (CAT) and glutathione reductase (GSSG-Red) activities. Despite these results, in vitro oxidative challenge studies with H2O2 demonstrated a greater susceptibility of liver to GSH depletion in the SHR, although no parallel change in thiobarbituric acid reactive substances (TBARS) production was observed. The comparatively lower plasma cholesterol observed in hypertensive SHR paralleled specific differences in liver catalase and glutathione redox antioxidant enzyme activities.     

Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter

oatp1 is an hepatic sinusoidal organic anion transporter that mediates uptake of various structurally unrelated organic compounds from blood. The driving force for uptake on oatp1 has not been identified, although a role for bicarbonate has recently been proposed. The present study examined whether oatp1-mediated uptake is energized by efflux (countertransport) of intracellular reduced glutathione (GSH), and whether hydrophobic glutathione S-conjugates such as leukotriene C4 (LTC4) and S-dinitrophenyl glutathione (DNP-SG) form a novel class of substrates for oatp1. Xenopus laevis oocytes injected with the complementary RNA for oapt1 demonstrated higher uptake of 10 nM [3H]LTC4 and 50 microM [3H]DNP-SG, and higher efflux of [3H]GSH (2.5 mM endogenous intracellular GSH concentration). The oatp1-stimulated LTC4 and DNP-SG uptake was independent of the Na+ gradient, cis-inhibited by known substrates of this transport protein and by 1 mM GSH, and was saturable, with apparent Km values of 0.27 +/- 0.06 and 408 +/- 95 microM, respectively. Uptake of [3H]taurocholate, an endogenous substrate of oatp1, was competitively inhibited by DNP-SG. Of significance, oatp1-mediated taurocholate and LTC4 uptake was cis-inhibited and trans-stimulated by GSH, and [3H]GSH efflux was enhanced in the presence of extracellular taurocholate or sulfobromophthalein, indicating that GSH efflux down its large electrochemical gradient provides the driving force for uptake via oatp1. The stoichiometry of GSH/taurocholate exchange was 1:1. These findings identify a new class of substrates for oatp1 and provide evidence for GSH-dependent oatp1-mediated substrate transport.     

Cloning, expression and characterisation of the cDNA encoding human hepatic squalene synthase, and its relationship to phytoene synthase

The reaction catalysed by squalene synthase (SQS) shows many similarities to that performed by another polyisoprene synthase, phytoene synthase (PhS). By identifying sequences conserved between yeast SQS (ySQS) and PhS, we have cloned a 2-kb cDNA (hSQS) encoding human SQS, a protein of 417 amino acids with a predicted M(r) of 48,041, which has only limited homology to ySQS. When expressed in E. coli, the hSQS cDNA directed the production of active enzyme. Two hSQS mRNA species of 2.0 and 1.55 kb have been identified which differ in their 3' untranslated sequences. The two mRNAs are present in roughly equal amounts in heart, placenta, lung, liver, kidney and pancreas, but the 2-kb mRNA predominates in brain and skeletal muscle. In HepG2 cells, both mRNAs are induced 2-4-fold by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin. In contrast, Northern blot analysis of rat tissues reveals only a 2.0-kb mRNA, which is considerably up-regulated in vivo by lovastatin.     

AdipoQ is a novel adipose-specific gene dysregulated in obesity

Adipose differentiation is accompanied by changes in cellular morphology, a dramatic accumulation of intracellular lipid and activation of a specific program of gene expression. Using an mRNA differential display technique, we have isolated a novel adipose cDNA, termed adipoQ. The adipoQ cDNA encodes a polypeptide of 247 amino acids with a secretory signal sequence at the amino terminus, a collagenous region (Gly-X-Y repeats), and a globular domain. The globular domain of adipoQ shares significant homology with subunits of complement factor C1q, collagen alpha 1(X), and the brain-specific factor cerebellin. The expression of adipoQ is highly specific to adipose tissue in both mouse and rat. Expression of adipoQ is observed exclusively in mature fat cells as the stromal-vascular fraction of fat tissue does not contain adipoQ mRNA. In cultured 3T3-F442A and 3T3-L1 preadipocytes, hormone-induced differentiation dramatically increases the level of expression for adipoQ. Furthermore, the expression of adipoQ mRNA is significantly reduced in the adipose tissues from obese mice and humans. Whereas the biological function of this polypeptide is presently unknown, the tissue-specific expression of a putative secreted protein suggests that this factor may function as a novel signaling molecule for adipose tissue.     

Disposition of the bromosulfophthalein-glutathione conjugate in the isolated perfused rat kidney

Renal elimination of the bromosulfophthalein-glutathione conjugate (BSP-GSH) after its i.v. administration in the rat in vivo is negligible. In our study we wanted to establish whether the high albumin-binding of BSP-GSH constitutes the major restrictive factor toward the urinary excretion of the compound. The renal disposition of BSP-GSH was studied in the isolated rat kidney during perfusions with or without albumin in the perfusate. The urinary clearance of BSP-GSH in the absence of albumin was very low (< 60 microliters/min) as compared to the inulin clearance (approximately 300 microliters/min). This indicates that albumin-binding is not the major reason for the low urinary clearance of BSP-GSH. Addition of albumin to the perfusate further decreased the urinary excretion by 60%. BSP-GSH is metabolized by the kidney into two major metabolites: the cysteinylglycine conjugate and the di-glutathione conjugate. Both metabolites appear in perfusate, which suggests that BSP-GSH undergoes tubular (re-)uptake. The di-glutathione conjugate is further metabolized to the di-cysteinylglycine conjugate. The di-glutathione conjugate and the di-cysteinylglycine conjugate are the major urinary components and the urinary elimination of BSP-GSH may depend on their formation. Inhibition of gamma-glutamyl transpeptidase activity with acivicin largely prevented the degradation to the cysteinylglycine and dicysteinylglycine conjugates of BSP. The total rate of urinary excretion, however, was only slightly lowered by acivicin. Apparently, cleavage of the gamma-glutamyl moiety is not relevant for the total urinary elimination of BSP-GSH.     

Microtubule-associated protein-2 in the rat testis: a novel site of expression

The testis is one of the most abundant sources of microtubule networks. These networks include mitotic and meiotic spindles, the spermatid manchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-specific protein with the capacity to bind tubulin, cAMP-dependent kinase, and calmodulin. MAP2 mRNA is processed into at least two variants encoding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of coding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deletion of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C- terminal regions of MAP2b. To determine whether MAP2 was present in the rat testis, microtubule preparations were isolated from adult rat testis and brain by means of taxol-mediated polymerization and analyzed by gel filtration, ELISA, and Western blotting using polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These results were confirmed by Northern blot analysis of total RNA from adult rat brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5-3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential to encode MAP2c was detected in enriched preparations of immature Sertoli cells and adult Leydig cells. Round spermatids contained at least two MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displayed a stage-specific pattern of expression. Immunohistochemistry showed a MAP2-like protein in both somatic and germ cells, with a particularly distinct localization within the cytoplasm of primary and secondary spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization of this protein was observed in the nucleus of many testicular cells.     

cDNA cloning and genomic organization of peroxisome proliferator-inducible long-chain acyl-CoA hydrolase from rat liver cytosol

The cDNA for a peroxisome proliferator-inducible long-chain acyl-CoA hydrolase from rat liver cytosol, referred to as rLACH2, was isolated and its genomic structure was determined. The cDNA encoded a 419-amino-acid polypeptide with a calculated molecular weight of 46,011. Sequence analysis identified an active-site serine motif (Gly-x-Ser-x-Gly) common to carboxylesterases and lipases. When expressed in Escherichia coli, the cDNA directed expression of a protein immunoreactive to an anti-rLACH2 antibody with a molecular mass of 47 kDa, identical to that of purified rLACH2. Northern blot analysis showed marked induction of rLACH2 mRNA in the liver after feeding rats with di(2-ethylhexyl)phthalate, a peroxisome proliferator. The rLACH2 gene spanned about 19 kb and comprised 3 exons, the intron/exon boundaries of which were consistent with the donor/acceptor splice rule. A putative peroxisome proliferator response element (AGGTCATGGTTCA) was identified in the 5'-flanking region, suggesting the involvement of peroxisome proliferator-activated receptors in the regulation of rLACH2 gene expression.