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J D McGivan

Publications and source records attributed to J D McGivan.

At least 19 recordsLinked to original sources
PubMed2006

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed2005

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.

Adipose Tissue↗
PubMed2001

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.

Amino Acid Transport System X-AG↗
PubMed1999

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.

ATP-Binding Cassette Transporters↗
PubMed1998

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.

Amino Acid Transport System X-AG↗
PubMed1997

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.

3T3 Cells↗
PubMed1996

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.

Amino Acid Transport System X-AG↗
PubMed1994

Amino acid deprivation-induced stress response in the bovine renal epithelial cell line NBL-1: induction of HSP 70 by phenylalanine.

Amino acid deprivation of the bovine renal epithelial cell line NBL-1 led to a range of responses by the heat shock and glucose regulated stress proteins. The classic heat shock induction of HSP 72 was found to be mimicked, without prior heat stress, by phenylalanine addition to cells simultaneously deprived of all other amino acids. Co-inclusion of alanine prevented the HSP 72 induction by phenylalanine but not that caused by heat stress. Phenylalanine also increased expression of HSP 70 mRNA in cells simultaneously deprived of other amino acids. The glucose regulated protein GRP 75 was increased upon amino acid deprivation. GRP94 was detectable in a 50 kDa form in control cells but was detected as a 94 kDa form upon amino acid deprivation which was further enhanced upon inclusion of phenylalanine. Addition of alanine to the starvation medium led to detection of the 50 kDa form only. Amino acid deprivation appears to mimic the glucose deprivation stress response. Inclusion of phenylalanine during amino acid deprivation leads to a stress response similar to that of heat shock in terms of HSP 72 induction. However, the two inducers are sensitive to different repression signals since only the phenylalanine-signal was subject to nihilation by alanine co-inclusion.

Amino Acids↗
PubMed1994

Regulation of glycosylation of Lamp-1 in the bovine renal epithelial cell line NBL-1 by changes in the concentration of extracellular phosphate.

We have identified the bovine renal homologue of Lamp-1 (lysosomal-associated membrane glycoprotein 1). It has very similar physical characteristics to other Lamp-1 proteins from a wide variety of tissues and species. Partial sequence analysis has shown it to be 61% identical with human Lamp-1 and about 50% identical with rat and mouse Lamp-1. The extent of glycosylation of bovine Lamp-1 alters in response to changes in the concentration of extracellular phosphate. Bovine renal epithelial cells (NBL-1) grown in normal or phosphate-starved medium contain Lamp-1 of 120 kDa. However, if cells are grown in medium containing 8-10 mM phosphate, they contain Lamp-1 of only 100 kDa. The core protein and mRNA levels have been shown to remain constant under both conditions. Therefore the only conclusion is that the extent of Lamp-1 glycosylation must be changing in response to the extracellular concentration of phosphate. Unlike Carlsson and Fukuda [(1990) J. Biol. Chem. 265, 20488-20495], who showed that the human Lamp-1 protein contained polylactosaminoglycan residues, we have been unable to demonstrate the partial deglycosylation of bovine Lamp-1 by endo-beta-galactosidase. This enzyme removes polylactosaminoglycan groups from glycoproteins, and therefore indicates that the carbohydrate structure of bovine Lamp-1 is probably different from that of other Lamp-1 proteins. At present the physiological importance of bovine renal Lamp-1 and the changes in its extent of glycosylation are unknown. In this paper we postulate that Lamp-1 may be involved in the cycling of plasma-membrane proteins to the lysosome. This is based on the finding that the only other known effect of high extracellular phosphate on NBL-1 cells is to cause a decrease in the Vmax. of plasma-membrane-associated Na(+)-dependent phosphate transport [Helps and McGivan (1991) Eur. J. Biochem. 200, 797-803].

Amino Acid Sequence↗
PubMed1994

Evidence for a regulatory protein involved in the increased activity of system A for neutral amino acid transport in osmotically stressed mammalian cells.

System A for neutral amino acid transport is increased by hypertonic shock in NBL-1 cells previously induced to express system A activity by amino acid starvation. The hypertonicity-mediated effect can be blocked by cycloheximide but is insensitive to tunicamycin. The activity induced may be inactivated irreversibly by the addition of system A substrates, by a rapid mechanism insensitive to cycloheximide. In CHO-K1 cells, hypertonicity increases system A activity, as has been shown in NBL-1 cells. This effect is additive to the activity produced by derepression of system A by amino acid starvation and is insensitive to tunicamycin. Furthermore, the alanine-resistant mutant CHO-K1 alar4, which bears a mutation affecting the regulatory gene R1, involved in the derepression of system A activity after amino acid starvation, is still able to respond to the hypertonic shock by increasing system A activity to a level similar to that described in hypertonicity-induced derepressed CHO-K1 (wild type) cells. These results suggest (i) that the hypertonicity-mediated increase of system A activity occurs through a mechanism other than that involved in system A derepression and (ii) that a regulatory protein coded by an osmotically sensitive gene is responsible for further activation of preexisting A carriers.

Amino Acids↗
PubMed1994

Regulation of System B0 amino-acid-transport activity in the renal epithelial cell line NBL-1 and concomitant changes in SAAT1 hybridizing transcripts.

alpha-(Methylamino)isobutyric acid (MeAIB) insensitive Na(+)-dependent alanine transport activity in the bovine kidney cell line NBL-1 was increased upon amino acid starvation (> or = 20% over control levels). When L-phenylalanine (3 mM) was included in the starvation medium the increase was further enhanced (> or = 85% over control levels). In cells grown in control medium the Vmax, for MeAIB-insensitive Na+/alanine co-transport was found to be 6.0 +/- 0.7 nmol/3 min per mg (Km 41 +/- 12 microM) and for L-phenylalanine-treated amino-acid-starved cells the Vmax. was 21 +/- 5 nmol/3 min per mg (Km 92 +/- 40 microM). The increase in Vmax. was prevented by cycloheximide. Substrate specificity analysis identified the L-phenylalanine-induced transport system as System B0. [35S]Methionine labelling of cells during the amino acid starvation/phenylalanine treatments resulted in the differential labelling of a protein of 78 kDa. Northern-blot analysis using a SAAT1-specific probe revealed the presence of a new transcript (3.2 kb) in RNA extracted from cells incubated in amino acid starvation medium with L-phenylalanine included. The present findings suggest a novel means of control for System B0 by the use of physiological stress. It is also proposed that SAAT1 and System-B0 transcripts have considerable sequence similarity.

Alanine↗
PubMed1994

Calreticulin--a stress protein induced in the renal epithelial cell line NBL-1 by amino acid deprivation.

Confluent monolayer cultures of the bovine kidney cell line NBL-1 were starved of amino acids in the presence of tracer concentrations of [35S]-methionine. Fluorographs of SDS-polyacrylamide gel separated membrane proteins revealed increased labelling of at least two proteins in starved cells relative to those in cells grown in complete medium. The patterns of Coomassie blue stained proteins from Concanavalin A-purified fractions of cells grown under fed and amino acid-starved conditions were similar but fluorography indicated the presence of one major labelled glycoprotein with a molecular weight of 62 kD in starved cells which was not present in fed cells. N-terminal amino acid analysis of the first 15 amino acids of the 62 kD protein and a protein of 60 kD found in control cells identified both proteins as calreticulin. N-terminal amino acid sequence analysis of a second amino acid starvation-up-regulated protein identified it as glucose-regulated protein GRP78. The amino acid sequences of calreticulin, GRP78 and two transport proteins known to be induced in amino acid starvation, have a common motif near the C-terminal end of the molecule. It is suggested that calreticulin is a member of a novel class of stress proteins induced by amino acid starvation.

Amino Acid Sequence↗