Breed-associated variations in the sequence of the pig 3beta-hydroxysteroid dehydrogenase gene.

The entire sequence of the pig 3beta-hy-droxysteroid dehydrogenase (3beta-HSD) gene has recently become known. This gene is deemed to be important in androstenone metabolism in pig liver, and its defective expression has been shown to be related to androstenone accumulation in adipose tissue and the development of boar taint. The aim of the present work was to do the following: 1) define the structure of the pig 3beta-HSD gene and 2) compare 3beta-HSD DNA sequences from pigs of different breeds, which vary in adipose tissue androstenone levels, with the purpose of identifying a polymorphism that might be responsible for differential 3beta-HSD expression. The 5'flanking and the coding region of 3beta-HSD were cloned and sequenced by conventional techniques. The 3beta-HSD coding regions were identical in pigs of different breeds and in animals with high and low androstenone levels. Significant sequence variations were found in the 5'flanking region of the 3beta-HSD gene, where differences in the number of TTAT repeats and 3 SNP were observed. The SNP were associated with the number of the TTAT repeats. These variations in the DNA sequence of the 3beta-HSD gene were not associated with the androstenone level in s.c. adipose tissue but were breed-dependent. The results of this work might be used for detection of the presence of Meishan genes in Western pig breeds, especially if the phenotype is not clearly established.

Relationship between the expression of hepatic but not testicular 3beta-hydroxysteroid dehydrogenase with androstenone deposition in pig adipose tissue.

This study investigated the relationship between expression of hepatic and testicular 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and accumulation of androstenone in adipose tissue because of its relation to boar taint. The experiments were performed on 13 Large White (50%) x Landrace (50%) and Meishan (25%) x Large White (25%) x Landrace (50%), pigs, which differed in the level of backfat androstenone. Our previous work showed that the major product of the hepatic androstenone metabolism is 3beta-androstenol. In this study, the formation of 3beta-androstenol was inhibited by the specific 3beta-HSD inhibitor trilostane. These results are the first direct confirmation that 3beta-HSD is the enzyme responsible for androstenone metabolism in the pig. The expression of the hepatic but not testicular 3beta-HSD protein showed a negative relationship with the level of backfat androstenone (r2 = 0.64; P < 0.001) and was accompanied by a reduced rate of the hepatic androstenone clearance. Low expression of 3beta-HSD protein in the liver of high androstenone pigs was also accompanied by a reduced level of 3beta-HSD mRNA (P < 0.001), which suggests a defective regulation of the hepatic 3beta-HSD expression at the level of transcription. In contrast, expression of the testicular 3beta-HSD protein did not differ between animals with high and low androstenone levels (P > 0.05) and was lower compared with the hepatic 3beta-HSD expression. Cloning and sequencing of the 3beta-HSD coding regions established that the hepatic and testicular 3beta-HSD cDNA have identical sequences, which were 98% similar to the human 3beta-HSD isoform I. It is suggested that expression of a single 3beta-HSD gene is regulated by different mechanisms in pig liver and testis. The liver-specific regulation of 3beta-HSD expression contributes to the low rate of hepatic androstenone metabolism and therefore can be considered as one of the factors regulating deposition of androstenone in pig adipose tissue and subsequent development of boar taint.

Cloning and mapping of the porcine cytochrome-p450 2E1 gene and its association with skatole levels in the domestic pig.

The porcine cytochrome-p450 2E1 (CYP2E1) gene was isolated by screening a pig BAC library and partially sequenced. This sequence information was used to identify six single nucleotide polymorphisms (SNPs) within the CYP2E1 gene and its promoter. In addition, a microsatellite marker tightly linked to the CYP2E1 gene was subcloned from the BAC. One of these markers was used to map the CYP2E1 gene distal of SWC27 on SSC14, well outside reported quantitative trait loci on SSC14 for skatole, indole and taste test measures of boar taint. However, in a population of commercial pigs scored for backfat skatole levels, there was evidence of association between a SNP in the CYP2E1 promoter and skatole deposition, although there was no significant association between this SNP and skatole levels in the experimental cross.

A new method of quantifying glutathione levels in freshly isolated single superfused rat cardiomyocytes.

INTRODUCTION: Glutathione (GSH) is an important antioxidant in the heart whose content changes during cardiac insults. However, there are currently no methods for continuously monitoring free cytoplasmic GSH levels in single isolated and superfused cardiomyocytes exposed to normal and pathological conditions. METHODS: GSH was measured using CellTracker Blue CMAC (Molecular Probes), a member of a new family of thiol-sensitive dyes. The fluorescence of 5 microM CellTracker Blue CMAC was measured in various solutions containing glutathione-S-transferase and in freshly isolated single superfused cardiomyocytes using an inverted fluorescence microscope. The cardiomyocytes were isolated by standard procedures and loaded with either CellTracker Blue CMAC or monochlorobimane by 15 min of shaking incubation in the dark at room temperature followed by centrifugation with resuspension of the cells in dye-free media. Cell volume was calculated from the 3H2O and [14C]sucrose space. RESULTS: CellTracker Blue CMAC fluorescence was linearly proportional to 0-100 microM GSH, as described by the equation: Y = 182.2 (X) + 681.6 (r2 = .99, P < .001). Fluorescence was not affected by changing the glutathione-S-transferase level, the calcium concentration, or the pH, neither was the fluorescence quenched by H2O2 or cyanide. Exposure of freshly isolated single superfused cardiomyocytes to oxidative stress in the presence of 0-1 mM H2O2 caused a progressive decrease in cellular GSH. In contrast, brief exposure to metabolic inhibition in the presence of 2.5 mM NaCN evoked a significant increase in cardiomyocyte GSH followed by a return to control levels during washoff. In comparison to monochlorobimane, cells loaded with CellTracker Blue CMAC gave a stronger signal with better cellular retention of the probe. DISCUSSION: These results suggest that CellTracker Blue CMAC fluorescence will be a good tool for measuring GSH in freshly isolated single superfused cardiomyocytes because it shows the expected changes to oxidative stress and metabolic inhibition, and is reversible.

Relationships between skatole and androstenone accumulation, and cytochrome P4502E1 expression in Meishan×Large White pigs.

The effects of age, castration and diet on accumulation of skatole and androstenone in the backfat of 50% Meishan male pigs, was studied in relation to boar taint. Skatole and androstenone deposition in backfat of entire males was increased with age (114, 144 and 174 days). Castration significantly decreased skatole and androstenone levels in all age groups. The level of cytochrome P4502E1, the initial enzyme involved in hepatic skatole metabolism, was increased by castration at all ages. These results suggest that the decrease in androstenone levels following castration affects the regulation of cytochrome P4502E1 expression, and as a consequence, results in a decrease in skatole levels. The use of sugar beet feed in the diet decreased the level of skatole and increased that of cytochrome P4502E1 expression, but did not affect androstenone levels. It is suggested that skatole accumulation is reduced both by castration and by diet via induction of cytochrome P4502E1. However, the mechanism of induction of cytochrome P4502E1 by diet is different from that involved in its induction by castration.

Characteristics of L-aspartate transport and expression of EAAC-1 in sarcolemmal vesicles and isolated cells from rat heart.

OBJECTIVE: L-Aspartate is an important intermediary metabolite in the heart and has also been implicated in myocardial protection, but little is known about its transport across the cardiac sarcolemma. In this study we have tested the hypothesis that the high affinity sodium-dependent aspartate transporter, EAAC-1 is expressed in heart and have also characterised aspartate transport into the myocardium. METHODS: Characteristics of L-[14C]aspartate uptake into rat heart were investigated using sarcolemmal vesicles and isolated myocytes. The expression of EAAC-1 in the two preparations was also investigated by western blotting. RESULTS: The K(m) and V(max) of L-aspartate uptake was 9.78+/-0.7 microM and 1.17+/-0.27 pmol/mg/s in vesicles compared to 6.53+/-1.24 microM and 13.65+/-1.0 pmol/microl/s in cells. In vesicles, L-aspartate uptake was dependent on external sodium and internal potassium, and was rheogenic. In cells, L-aspartate uptake was also dependent on external sodium. Addition of unlabelled L- and D-aspartate and L-glutamate significantly inhibited L-[14C]aspartate uptake in both preparations but D-glutamate had no effect. An antibody to the aspartate transporter, EAAC-1 recognised a protein of appropriate size in both vesicles and cells. CONCLUSIONS: L-aspartate uptake in heart is mediated by a high affinity sodium-dependent transporter. This is accompanied by the expression in heart of EAAC-1. The physiological significance of this transporter with respect to aspartate utilisation in the heart is discussed.

The rat hepatoma cell line H4-II-E-C3 expresses high activities of the high-affinity glutamate transporter GLT-1A.

The rate of uptake of aspartate into the rat hepatoma cell line H4-II-E-C3 is very much higher than that exhibited by normal rat hepatocytes. Using an RT-PCR-based strategy, a glutamate transporter resembling mouse liver GLT-1A has been cloned from H4-II-E-C3 cells. Northern blotting confirmed that relatively high levels of mRNA for GLT-1A are expressed in hepatoma cells compared with negligible levels in rat hepatocytes. To our knowledge, this is the first report of the cloning of a high-affinity glutamate transporter from a transformed cell line and also the first demonstration of functional expression of GLT-1 outside the central nervous system.

Regulation of high-affinity glutamate transport by amino acid deprivation and hyperosmotic stress.

High-affinity glutamate transport activity is induced by stress in NBL-1 cells. Exposure of cells to hyperosmotic medium led to an induction of the EAAC1 glutamate transporter, preceded by a large increase in EAAC1 mRNA levels. Culture of cells in amino acid-free medium also caused a protein synthesis-dependent increase in glutamate transport activity, but this was not accompanied by an increase of either EAAC1 mRNA or protein. Indirect evidence suggests that the increase in EAAC1 activity in the latter case may be due to the synthesis of an activator protein in response to decreased intracellular glutamate concentrations.

Rat hepatoma cells express novel transport systems for glutamine and glutamate in addition to those present in normal rat hepatocytes.

The rat hepatoma cell line H4-II-E was found to express much higher activities of Na+-dependent glutamine and aspartate transport than those observed in normal cultured hepatocytes, in agreement with previous work of others on human hepatocytes. Na+-dependent glutamine transport in rat hepatoma cells could be resolved into two components. One was pH-dependent, tolerated Li+ for Na+ substitution and was inhibited only by asparagine and histidine; characteristics similar to those of transport System N in hepatocytes. The other transport system had a similar Km for glutamine but was pH independent, did not accept Li+ ions and was completely inhibited by excess concentrations of lysine, histidine, leucine, serine and cysteine, but not by methyl-aminoisobutyrate or phenylalanine. This pattern of inhibition is distinct from that of any transporter occurring in normal hepatocytes and may indicate the presence of a new transporter isoform. Similar results were obtained with the cell line HTC. Na+-dependent aspartate transport in H4 hepatoma cells was mediated by a high-affinity system (Km 5 microM) and was inhibited by D-aspartate and L-glutamate but not by d-glutamate-properties characteristic of the high-affinity glutamate transporter EAAC1. C-terminal antibodies to the EAAC1 protein recognized a single band of 58 kDa in hepatocyte membranes, but an additional strong band of 60 kDa was present in H4 hepatoma cells. These results provide further evidence for the view that tumour cells may express additional isoforms of amino acid transport systems which are not present in non-transformed cells.

Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells.

The role of calreticulin as a stress-induced molecular chaperone protein of the endoplasmic reticulum is becoming more apparent. We characterize here the induction of calreticulin in response to complete amino acid deprivation in Chinese hamster ovary cells. Amino acid deprivation caused a 4-fold increase in calreticulin protein levels over a period of 4-10 h. In addition to an overall increase in protein levels, the glycosylation of calreticulin was increased. This glycosylation event was blocked by tunicamycin and was not required for the increase in calreticulin protein levels. Immunofluorescence studies localized calreticulin to the ER of CHO cells, and no significant change was observed after amino acid deprivation. Northern-blot analysis showed that calreticulin mRNA levels were increased approx. 10-fold in response to complete amino acid deprivation. The response was sensitive to actinomycin D and alpha-amanitin, implying that regulation is primarily at the level of transcription. These results are similar to the large increases in asparagine synthetase mRNA observed in response to amino acid deprivation, but the amino acid-deprivation-response element identified to be involved in asparagine synthetase induction is absent from the calreticulin promoter.

Induction of the stress protein Grp75 by amino acid deprivation in CHO cells does not involve an increase in Grp75 mRNA levels.

The induction of the stress protein Grp75 in response to amino acid deprivation of Chinese Hamster Ovary cells was characterised using a specific monoclonal antibody. A 2-fold increase in the Grp75 protein content occurred over a period of 5-10 h after incubation of the cells in amino acid-free medium. A partial induction was obtained when either all non-essential amino acids or all essential amino acids were omitted from the medium indicating a broad-specificity response. Deletion of the single amino acids tryptophan, histidine or phenylalanine from otherwise complete medium also produced a partial induction of the protein. The increase in the level of Grp75 was completely blocked by cycloheximide, but only partially blocked by the inhibitors of mRNA synthesis actinomycin D and alpha-amanitin. A specific cDNA probe for Grp75 was generated by PCR and used to quantify mRNA levels. No increase in Grp75 mRNA was observed during the induction of the protein indicating that the primary regulation of Grp75 expression was not at the transcriptional level. These results contrast with the large increase in asparagine synthetase mRNA which has been shown to occur during amino acid deprivation, and indicate that cells respond to this form of stress by more than one mechanism.

Identification and partial characterization of a novel membrane glycoprotein induced by amino acid deprivation in renal epithelial cells.

We have identified a protein of 110 kDa in the renal epithelial cell line NBL-1. which is induced on incubation of the cells in an amino-acid-free medium. The protein was purified on conA-Sepharose and subjected to N-terminal sequencing. The sequence obtained. VDRINFKT, does not correspond to any protein in the databases. Antipeptide antibodies made to this sequence recognised a single protein of 110 kDa in whole cell membranes and in a conconavalin A protein extract. Using the antibody on Western blots, the protein was induced 2.5-3 fold in 10-15 h and the induction was inhibited by cycloheximide and tunicamycin. The protein was found also in rat liver plasma membranes. A procedure for the partial purification of this protein from rat liver is described, and some internal sequence is reported. The possible relationship of the induction of this novel protein to the induction of amino acid transport in these cells by amino acid deprivation is discussed.

Induction of high affinity glutamate transport activity by amino acid deprivation in renal epithelial cells does not involve an increase in the amount of transporter protein.

In renal epithelial cells amino acid deprivation induces an increase in L-Asp transport with a doubling of the Vmax and no change in Km (4.5 micronM) in a cycloheximide-sensitive process. The induction of sodium-depending L-aspartate transport was inhibited by single amino acids that are metabolized to produce glutamate but not by those that do not produce glutamate. The transaminase inhibitor aminooxyacetate in glutamine-free medium caused a decrease in cell glutamate content and an induction of glutamate transport. In complete medium aminooxyacetate neither decreased cell glutamate nor increased transport activity. These results are consistent with a triggering of induction of transport by low intracellular glutamate concentrations. High affinity glutamate transport in these cells is mediated by the excitatory amino acid carrier 1 (EAAC1) gene product. Western blotting using antibodies to the C-terminal region of EAAC1 showed that there is no increase in the amount of EAAC1 protein on prolonged incubation in amino acid-free medium. Conversely, the induction of high affinity glutamate transport by hyperosmotic shock was accompanied by an increase in EAAC1 protein. It is proposed that low glutamate levels lead to the induction of a putative protein that activates the EAAC1 transporter. A model illustrating such a mechanism is described.

Regulation of amino acid transport in the renal epithelial cell line NBL-1.

The activities of the transport systems A, B° and XAG- are induced by various forms of stress in renal epithelial cells. Amino acid deprivation induces System A and XAG- in a protein-synthesis dependent process. In the case of System XAG- evidence is presented that induction of transport does not involve an increase in the amount of mRNA for the transporter or of the amount of transport protein. Preliminary evidence for the existence of a novel glycoprotein which is induced in parallel to the induction of these transport systems is presented. It is suggested that the induction of amino acid transport proteins and of some of the so-called stress proteins may be triggered by a common molecular mechanism.

Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself.

Cloning, sequence analysis and expression of the cDNA encoding a sodium-dependent phosphate transporter from the bovine renal epithelial cell line NBL-1.

We have isolated a full-length cDNA clone of 2.2 kb from a lambda ZapII NBL-1 (bovine renal epithelial cell) cDNA library using a portion of the rat renal sodium-dependent phosphate transporter cDNA as a probe. Expression of this cDNA clone in the COS cell line has shown it to specifically encode a sodium-dependent phosphate transporter from bovine renal epithelial cells. Sequence analysis of the clone showed a single open reading frame of 693 amino acids which has 70% similarity to the phosphate transporter of rat and human kidney [Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J. & Murer, H. (1993) Proc. Natl Acad. Sci. USA 90, 5979-5983]. Hydropathy plots of the derived amino acid sequence show at least eight possible transmembrane regions, again in agreement with data for the rat and human transporters. The sequence contains nine putative N-glycosylation sites and nine potential sites for protein kinase C phosphorylation. Previously we have shown that the kinetics of phosphate transport into NBL-1 cells are significantly different to those for opossum kidney cells or rat kidney [Helps, C. & McGivan, J. (1991) Eur. J. Biochem. 200, 797-803]. This difference is presumably related to differences in the amino acid sequence between this protein and other cloned phosphate transporters.