Glutathione and glutathione conjugate efflux from cultured liver cells

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C₁ and the -glutamyl transpeptidase containing, tumorigenic ARL-16T₂, has been assessed under basal condition and during chronic treatment with 75 and 150 M ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines.Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 M EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of -glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T₂ cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T₂ cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased in racellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GS-EA the glutathione conjugate of ethacrynic acid - EA ethacrynic acid - CDNB 1-chloro 2,4-dinitrobenzene - HBS HEPES buffered saline - OTC 2-L-oxothiazolidine 4-carboxylic acid - CYSSG cysteinyl-glutathione mixed disulfide - FDNB 1-fluoro-2,4-dinitrobenzene - GCS -glutamyl cysteine synthetase - GST glutathione-S-transferase - BCA bicinchoninic acid - SDS sodium dodecyl sulfate - PCA perchloric acid     

Rat hepatocytes prepared without collagenase: Prolonged retention of differentiated characteristics in culture

Rat hepatocytes prepared by collagenase digestion or ED TA dissociation were examined in culture for comparison of culture stability and morphology, and retention of selected adult rat liver characteristics. Cells prepared by EDTA perfusion followed by Percoll cen trifugation were deemed to form confluent monolayer cultures more rapidly and monolayers remained intact for up to 21 days without signs of nonparenchymal cell growth or loss of primary hepatocyte appearance. The spectrally determined cytochrome P-450 content remained constant through eight days in culture. Collagenase prepared cells contained an identical amount of P-450 but within 72 hr lost greater than 80% of the spectrally detectable P-450. Glutathione (GSH) content was higher in the EDTA-prepared hepatocytes and remained constant with only a modest effect of transferrin and selenium (TI S) supplementation, while GSH levels in collagenaseprepared cells increased, thereafter decreased with time in culture and was dependent on TI S supplementation. Cells prepared with ED TA also displayed an increase in GSH efflux rate in response to chronic GSH depletion by ethacrynic acid. -Cystathionase (CNase) activity was retained at initial levels in ED TAprepared hepatocytes supplemented with TI S and declined only about 25% in unsupplemented cells. Collagenase-prepared cells lost 75% of CNase activity by 72 hr. The established marker of hepatocyte neoplastic transformation, -glutamyl trans-peptidase (GGT), increased rapidly in collagenase-prepared cells. The accumulation of GGT was slowed by T/S supplementation. GGT activity did not increase in EDTA-prepared hepatocytes. Evaluation of morphological and biochemical criteria suggest that hepatocytes prepared without collagenase present superior model systems for the study of biochemical events through more extended culture times.Abbreviations CNase -cystathionase - EDTA ethylenediamine tetraacetic acid - GGT -glutamyl transpeptidase - GSH reduced glutathione - GSSG oxidized glutathione - HEPES N-2-hydroxyethylpiperizine-N-2-ethane-sulfonic acid - SAH S-adenosylhomocysteine - SAM S-adenosylmethionine - T/S transferrin- and selenite-supplemented     

Metabolism of leukotrienes

Summary The in vitro metabolism of leukotriene B₄ is initiated by -hydroxylation. This reaction is followed by oxidation of the -hydroxyl group to a carboxyl group. In vivo extensive -oxidation occurs and the main excreted products after administration of leukotriene B₄ are water and carbon dioxide.Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal y-glutamyl residue, catalyzed by the enzyme -glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E₄, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C₄. In rats, the two major fecal metabolities of leukotriene C₄4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E₄. These compounds are formed in reactions between leukotriene E₄ or 11-trans leukotriene E₄ and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.     

Influence of selenium on glutathione and some associated enzymes in rats with mammary tumor induced by 7,12-Dimethylbenz(a)anthracene

A recent finding in epidemiological and laboratory studies suggests that the ratio of selenium to glutathione is lower in breast cancer subjects than its control counterparts. Selenium, an antioxidant and anticarcinogen, can modify the status of glutathione and some associated enzymes by blocking peroxidation of lipids in membranes of cancer subjects. Studies were conducted using female albino rats of Wistar strain bearing mammary tumor induced by 7,12-dimethylbenz(a) anthracene to assess the biological role of selenium on some antioxidant enzymes associated with the maintenance of glutathione status. For induction of mammary tumor, 25 mg DMBA in a 1 ml emulsion of sunflower oil and physiological saline was injected subcutaneously to each rat. One group in each of control and tumor bearing rats, were fed 5 mg sodium selenite/kg diet from the day of tumor induction for 24 weeks. Increase in the reduced glutathione concentration was preceded by significant increase in the oxidized glutathione as well as in the activities of -glutamylcysteine synthetase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase by selenium administration in rats bearing tumor. However, selenium administration to rats bearing tumor decreased the activity of -glutamyl transpeptidase. These observations clearly demonstrate the influence of dietary selenium supplementation in correcting abnormal changes in glutathione turnover and some associated enzymes in tumor induced rats.     

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l^–1 with a productivity of 1 g l^–1 h^–1 by pulsed-feeding of citric acid (1.44 g h^–1) and l-glutamic acid (2.4 g h^–1) when citric acid was depleted from the culture medium.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

Identification of high molecular weight antigens structurally related to gamma-glutamyl transferase in epithelial tissues

Summary Heterologous antibodies to gamma-glutamyl transferase (GT), an ectoenzyme associated with the apical surface of many types of epithelial cells involved in secretion and transport, have been used to identify and partially characterize the spectrum of antigens in a series of epithelial tissues that exhibit a range of enzyme activities. In addition to antigens corresponding to the subunits of the active enzyme (mol wt 55K, 30K), antigens of mol wt 85–95K have been detected using an antibody raised against the enzyme purified in nonionic detergent. The latter species are shown to share antigenic determinants with and to be structurally related to the enzyme subunits; however, they do not bind significantly to antibodies raised to protease-solubilized GT. Further, they constitute the major antigens in tissues that exhibit relatively low levels of enzyme activity. These polypeptides are apparently larger than a recently characterized biosynthetic precursor of the GT subunits. Although they do not have GT activity themselves and their function is undefined, the possibility that they may represent highly glycosylated polypeptides related either to GT precursors (that persist without processing) or to the large enzyme subunit merits consideration.     

Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Different pattern of T cell receptor δ gene rearrangement in tumour-infiltrating lymphocytes and peripheral blood in patients with solid tumours

Tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) from four patients with renal-cell carcinoma (three paired with blood), two colon carcinomas (both paired with blood) and two melanomas (blood was not available) were analysed for the T cell receptor (TCR) gene repertoire. Polymerase chain reaction analysis, employing a panel of specific primers for TCR gene segments, showed different gene rearrangement patterns in TIL and PBL in all patients. Simultaneous analysis of TIL and PBL revealed the presence of lymphoid cells in the tumour tissue that were not present in the periphery. These results demonstrate that, although tumor-infiltrating lymphocytes contain / T cells within the range observed in peripheral blood, these cells differ from those in peripheral blood in their gene repertoire and this may account for selective accumulation or/and in situ amplification of / lymphocytes at the tumour site, indicating a unique type of host reaction against tumors.     

Oxidative Stress Promotes the Regression of Fetal Liver Hemopoiesis

Although apoptosis is believed to play an important role in the ontogenetic development of animals, the molecular mechanism that triggers the regression of liver hemopoiesis during the perinatal period is not known. Apoptosis is induced by many factors such as a decrease in growth factors and increased oxygen stress. Since hepatic gamma-glutamyl transferase (GT) levels change markedly during the perinatal period in rodents, the metabolism of glutathione (GSH), a naturally occurring major antioxidant, might change significantly in and around liver cells. Hemopoietic cells but not hepatocytes exhibit significant apoptosis in thiol-free medium and the hemopoietic apoptosis can be inhibited by various thiols, such as L-cysteine, N-acetyl-L-cysteine, and GSH. The contribution of GSH levels in and around fetal liver cells in the triggering of apoptosis in hemopoietic cells is discussed.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Analysis of the pattern of gene expression during human adipogenesis by DNA microarray

High density DNA microarrays containing over 5000 cDNA clones were used to carry out a comprehensive investigation of gene expression during adipogenesis. Complex probes synthesized from total RNA were hybridized to the arrays to determine the level of mRNA expression of each arrayed gene. Thirty three genes (29 known and 4 ESTs with no identified homologies) have been found to alter their level of expression more than 2.5-fold after differentiation. The quantitative measurement by DNA array was in good agreement with conventional Northern blot analysis of selected genes. Our results demonstrate that utilization of a DNA array is a speedy, efficient and quantitative approach to profile the expression of a large number of genes.     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation

Myristoylated G_o was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_o) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_o, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPS) activation induced disaggregation of the G_o oligomers to monomers. The G_o was crosslinked by a cross-linker, N,N"-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_o was pretreated with GTPS. Immunoblot analysis also demonstrated oligomerization of the purified G_o proteins and its disaggregation triggered by GTPS. These results provided direct evidence for the disaggregation–coupling theory and the disaggregation action of GTPS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.     

Effect of interferon γ on the sensitivity of bovine-papilloma-virus(BPV1)-transformed cell lines to cell-mediated cytotoxicity

Summary The effect of interferon (IFN) on the immunogenicity and immunosensitivity of mouse cell lines transformed by bovine papillomavirus type 1 (BPV1) DNA was examined in a syngeneic mouse model. The overnight incubation of BPV1-transformed cell lines with 100 IU/ml IFN did not affect their ability to induce the generation of cytotoxic effector cells but it clearly increased their sensitivity to lysis by interleukin-2-induced lymphokine-activated killer (LAK) cells and by nonspecific LAK-type effector cells induced by BPV-1-transformed cell lines. The treatment of two allogeneic lymphoid tumour cell lines, P815X2 and YAC-1, with IFN either decreased or had no effect on their sensitivity to LAK-cell-mediated lysis.