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D A Rice

Publications and source records attributed to D A Rice.

At least 37 records · Page 2Linked to original sources
PubMed1990

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit.

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.

Animals↗
PubMed1990

Survey of levels of propetamphos and diazinon used to control sheep scab in Northern Ireland.

As a result of an increase in the incidence of sheep scab in Northern Ireland, the concentrations of propetamphos and diazinon were measured during 1987 and 1988 in fleece and liquid dip samples from selected flocks, including some in which inadequate dipping was suspected. Sixty-five per cent and 68 per cent of the liquid dip samples contained less than the manufacturer's recommended maintenance concentrations for propetamphos and diazinon respectively. The concentrations found in fleece were also lower than those found in sheep which were dipped with the recommended concentrations of propetamphos and diazinon in a controlled experiment.

Animals↗
PubMed1990

Development of a specific radioimmunoassay for vitamin B12 and its application in the diagnosis of cobalt deficiency in sheep.

A radioimmunoassay (RIA) for vitamin B12 is described. Antisera were raised in rabbits using a conjugate between 5'-O-succinyl cyanocobalamin and chicken serum albumin. The antibody is largely specific for the beta (upper) face of the vitamin B12 molecule. The RIA can detect vitamin B12 down to concentrations of 37 pmol/L and correlated well (r = 0.980, p less than 0.001) with a commercial radioassay. The use of this RIA, together with an assay for plasma methylmalonic acid, in the diagnosis of cobalt/vitamin B12 deficiency in sheep is described.

Animals↗
PubMed1990

Identification and characterization of two upstream elements that regulate adrenocortical expression of steroid 11 beta-hydroxylase.

We have studied the mechanisms that regulate the expression of the mouse gene encoding steroid 11 beta-hydroxylase (11 beta-OHase), a steroidogenic cytochrome P450 enzyme that is expressed only in the adrenal cortex. DNase I footprinting and gel-mobility shift analyses revealed potential regulatory elements at -370 and -310 in the 11 beta-OHase promoter region. To determine the contributions of these elements to expression, we altered their sequences by site-selected mutagenesis and studied promoter activity after transfection into Y1 mouse adrenocortical tumor cells. Mutation of either element markedly decreased basal promoter activity but did not affect the response to treatment with 8-bromo cAMP. These experiments thus document the functional roles of these elements, within the context of the intact promoter, in constitutive expression of 11 beta-OHase. Moreover, addition of either of these elements to p-40GH, a 5'-deletion plasmid containing 11 beta-OHase sequences from -40 to +8 upstream of a growth hormone reporter gene, significantly increased promoter activity but did not confer cAMP responsiveness. Finally, increased expression was seen after transfection of Y1 derivatives deficient in cAMP-dependent protein kinase, indicating that neither element required cAMP-dependent protein kinase activity. These studies thus define two regulatory elements that play important roles in 11 beta-OHase expression.

Adrenal Cortex Neoplasms↗
PubMed1989

Site-directed mutagenesis of a conserved, extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase.

Site-specific mutagenesis was used to study the function of a conserved, extracellular aspartic acid residue from the sheep Na,K-ATPase alpha subunit. This amino acid, Asp-121, is the penultimate residue of the first extracellular domain of the alpha subunit. The border residues of this particular extracellular loop of the alpha subunit have been shown to be determinants of ouabain sensitivity (Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to determine if Asp-121 is involved in ouabain binding, five different amino acid substitutions at this position were generated. Four of the five mutant alpha subunits, containing either Asn, Ala, Glu, or Ser in place of Asp-121, conferred ouabain resistance to HeLa cells when expressed in those cells. Cloned sublines of cells selected in ouabain were characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. The cells expressing the mutant Na,K-ATPase alpha subunit containing either Asn, Ala, Glu, or Ser in place of Asp-121 contained a component of Na,K-ATPase activity that was nearly 100-times more resistant to ouabain than the endogenous HeLa (human) or sheep enzyme. Apparently, conservative (Glu for Asp), isosteric (Asn for Asp), and nonconservative (Ala or Ser for Asp) substitutions all significantly decreased ouabain sensitivity. These data suggest that Asp-121 of the sheep Na,K-ATPase alpha subunit participates in the binding interaction between the enzyme and ouabain.

Amino Acid Sequence↗
PubMed1989

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene.

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.

Adrenal Cortex Neoplasms↗
PubMed1989

The roles of cAMP and cAMP-dependent protein kinase in the expression of cholesterol side chain cleavage and steroid 11 beta-hydroxylase genes in mouse adrenocortical tumor cells.

The expression of the genes encoding cholesterol side chain cleavage enzyme (SCC) and steroid 11 beta-hydroxylase (11 beta-OHase) was examined in Y1 mouse adrenocortical tumor cells and in derivative cell lines defective in cAMP-dependent protein kinase activity. Y1 cells expressed both genes constitutively, and treatment with 8-bromo-cAMP (8-Br-cAMP) increased expression 5-10-fold. In three independent protein kinase mutants, expression of SCC and 11 beta-OHase was impaired to degrees dependent upon the severity of defect in cAMP-dependent protein kinase activity. In Kin-2, the least impaired mutant clone, basal expression of SCC was the same as in Y1 cells. Treatment of Kin-2 with 8-Br-cAMP increased SCC RNA to the levels seen in stimulated Y1 cells. In contrast, clone Kin-8, the most severe mutant, expressed markedly diminished basal and 8-Br-cAMP-stimulated levels of SCC mRNA. Kin-7 had basal and 8-Br-cAMP-stimulated levels of SCC mRNA which were intermediate to Kin-2 and Kin-8. None of the Kin mutants constitutively expressed detectable levels of 11 beta-OHase transcripts, and only Kin-2 responded to treatment with 8-Br-cAMP with increased expression of 11 beta-OHase; however, the time course of induction in Kin-2 was significantly delayed. The disparate patterns of expression of SCC and 11 beta-OHase in the Kin mutants suggest that these genes differ in their absolute requirement for cAMP-dependent protein kinase activity. Experiments also were performed in which Kin-7 and Kin-8 mutants were restored to cAMP-responsive states by transfection with genes encoding normal sub-units of cAMP-dependent protein kinase. These phenotypic revertants recovered 8-Br-cAMP-inducible expression of SCC and 11 beta-OHase. These results strongly support the hypothesis that impaired expression of steroidogenic enzymes in the Kin mutants results directly from defects in cAMP-dependent protein kinase activity.

Adrenal Cortex Neoplasms↗
PubMed1989

Structural and functional analysis of the promoter region of the gene encoding mouse steroid 11 beta-hydroxylase.

The mouse gene encoding adrenal steroid 11 beta-hydroxylase (11 beta-OHase) has been cloned and the nucleotide sequence of its 5' end has been determined. The coding regions sequenced are homologous (75%) to the sequence of bovine 11 beta-OHase cDNA. The 5'-flanking region of the 11 beta-OHase gene contains a potential cAMP response element (TGACGTGA) located 56 base pairs upstream of the transcription initiation site (position -56) and two motifs at positions -249 and -148 which are similar to an element postulated to be required for the expression of 21-hydroxylase. Transfection of mouse Y1 adrenocortical tumor cells and MA-10 testicular Leydig cells with plasmids containing the 11 beta-OHase promoter linked to a growth hormone reporter gene showed that the 11 beta-OHase promoter can direct cell-specific expression. Deletion analyses of the 5'-flanking region suggest that multiple sequence elements, one of which is located between positions -425 and -338 and a second between positions -338 and -123, interact to produce full levels of promoter activity. Mutant Y1 cells defective in cAMP-dependent protein kinase activity do not express growth hormone driven by the 11 beta-OHase promoter, indicating that expression of 11 beta-OHase in Y1 cells requires an intact cAMP second messenger system. Moreover, mutation of the putative cAMP response element at position -56 abolishes expression. These experiments thus present a useful system for the investigation of cis-acting elements involved in the transcriptional regulation of 11 beta-OHase.

Amino Acid Sequence↗
PubMed1989

Sequential changes in plasma methylmalonic acid and vitamin B12 in sheep eating cobalt-deficient grass.

Methylmalonic acid (MMA) concentrations are elevated in plasma as a result of vitamin B12 deficiency. This study reports the sequential changes in plasma MMA in lambs maintained on a cobalt-deficient pasture compared with supplemented controls. The results indicate that MMA is elevated in the early stages of deficiency, preceding the onset of loss of production and clinical signs of disease. It remains elevated as long as the lambs are unsupplemented with cobalt (Co). The most striking clinical sign was a loss of body condition as opposed to weight. The defect in the methylmalonyl CoA mutase is obviously an early defect in cobalt deficiency.

Animals↗
PubMed1989

Determination of chlortetracycline residues in tissues using high-performance liquid chromatography with fluorescence detection.

A method is described for the determination of chlortetracycline residues in tissue samples. The samples were extracted into a hydrochloric acid - glycine solution and the extracts concentrated and purified on cyclohexyl-bonded reversed-phase cartridges. Any chlortetracycline present was converted to iso-chlortetracycline at pH 12, which was then separated from interfering compounds on a reversed-phase polymeric column using high-performance liquid chromatography with fluorescence detection. The detection and determination limits of the assay were 20 and 50 ng g-1, respectively, making it suitable for statutory residue testing purposes.

Chlortetracycline↗
PubMed1989

Methylmalonic acid in the diagnosis of cobalt deficiency in barley-fed lambs.

Eight lambs were fed on a cobalt-deficient whole-barley diet supplemented with urea, vitamins and minerals. Four control lambs were fed on the same diet which had been further supplemented with Co. Plasma vitamin B12 levels in the Co-depleted group declined rapidly, falling below the normal range within 5 weeks. Differences between the live weights of the animals in the two groups approached statistical significance by week 14. However, methylmalonic acid (MMA) rose above normal levels in the Co-depleted group within 7 weeks. This suggested that an elevated plasma concentration of MMA is a comparatively early indicator of functional vitamin B12 deficiency. It is recommended that 10 mumol/l be the upper level of normality for plasma MMA concentration in barley-fed animals, in contrast with the level of 5 mumol/l for grass-fed animals. Changes in the plasma concentrations of MMA and ethylmalonic acid associated with feeding the barley-based diet per se did not significantly affect the validity of the gas-liquid chromatographic assay for MMA.

Animals↗
PubMed1989

Regulation of 21-hydroxylase gene expression.

The steroid 21-hydroxylase (21-OH) gene is selectively expressed in the adrenal cortex and is transcriptionally regulated by ACTH. We examined the role of the 5'-flanking sequences of 21-OH in this regulated expression by analyzing their ability to direct the expression of a human growth hormone (hGH) reporter gene upon transfection into Y1 mouse adrenocortical tumor cells. The 330 bp of 5'-flanking sequences directed basal and hormonally-inducible expression of hGH in Y1 cells, but did not direct expression in I-10 mouse testicular Leydig cells. Both constitutive and hormonally-inducible expression required a functional cAMP-dependent protein kinase. These results indicate that the first 330 bp of 5'-flanking sequences of the 21-OH gene contain sufficient information for cell-specific and hormonally regulated expression, and that this expression requires the integrity of cAMP-dependent protein kinase. Markedly lower expression of hGH was seen when 156 bp of 5'-flanking sequences were placed in front of the reporter gene, suggesting that sequences between -330 and -156 are essential for expression. The addition of sequences from -330 to -150 to the p-156GH plasmid, in either the correct or the reverse orientation, restored promoter activity to approximately the level obtained with the 330 bp of 5'-flanking sequences. Moreover, the addition of sequences from -230 to -150 increased by 5-fold the expression of hGH driven by the heterologous thymidine kinase promoter. Based on these results, we conclude that an enhancer element is contained within the sequences from 230 to 150 bp upstream of the transcription initiation site.

Adrenal Cortex Neoplasms↗
PubMed1989

Vitamin E, selenium, and polyunsaturated fatty acid concentrations and glutathione peroxidase activity in tissues from pigs with dietetic microangiopathy (mulberry heart disease).

Tissues of 27 pigs with spontaneous dietetic microangiopathy (DM) and of 27 control pigs that died of causes unrelated to vitamin E and selenium (E-Se) deficiency were analyzed for alpha-tocopherol, Se and polyunsaturated fatty acid (PUFA) concentrations and for glutathione perioxidase (GSH-Px) activity. These variables (except for GSH-Px) also were measured in rations fed to control pigs and pigs with DM. Swine with DM had lower heart and liver alpha-tocopherol concentrations than did control pigs. Heart and kidney Se concentrations and heart and liver PUFA concentrations were similar in pigs of either group. Diets fed to both groups of pigs contained similar content of alpha-tocopherol, Se, oil, and PUFA; alpha-tocopherol and Se concentrations in the diets of both groups of pigs were high. In spite of apparently adequate amount of dietary alpha-tocopherol, results indicate that pigs with DM had lower tissue alpha-tocopherol concentration than did control pigs. Spontaneous DM is associated with altered alpha-tocopherol metabolism, but is unrelated to alterations in dietary Se and PUFA contents and tissue Se and PUFA concentrations and GSH-Px activity.

Animals↗
PubMed1988

Selective morphologic alterations of the cardiac conduction system in calves deficient in vitamin E and selenium.

Vitamin E and selenium (E-Se) deficiency causes necrosis of the contractile myocardium in many species but does not usually affect the cells of the conduction system. In the present study, experimental E-Se deficiency in cattle produced preferential degeneration and necrosis of Purkinje cardiocytes. Calves fed deficient diets for 127-137 days had sublethal damage characterized histologically by sarcoplasmic accumulation of lipopigment granules; ultrastructurally, these granules corresponded to cytolysosomes that had a heterogeneous ultrastructure. Alterations in necrotic cells included mitochondrial mineralization, sarcoplasmic condensation, and plasmalemmal fragmentation. Necrosis of Purkinje cells was followed by macrophagic penetration of the external lamina, phagocytosis of necrotic sarcoplasm, and repair by fibrosis. Furthermore, E-Se depletion of calves resulted in only minimal alterations in the contractile myocardium. In contrast, feeding supplements of polyunsaturated fatty acids to E-Se-deficient calves intensified the Purkinje cell damage but also resulted in widespread degeneration and necrosis of the contractile myocardium. Accumulation of lipopigment supports a pathogenetic role for lipoperoxidation in development of the cardiac lesions of E-Se deficiency. These lesions constitute a unique example of preferential damage to Purkinje cardiocytes. This model offers an attractive method of studying damage and repair to the cardiac conduction system.

Animals↗
PubMed1987

Evaluation of the efficacy of trace element supplementation of an anthelmintic.

The efficacy of the selenium and cobalt contents of a trace element supplemented anthelmintic was evaluated in a series of trials with lambs in Scotland and Northern Ireland. The product was compared with other methods of supplementation and was found to be an adequate source of selenium when administered at monthly intervals. Its cobalt content was shown to be of some nutritional benefit on four of five sites but not as effective as the monthly 250 mg oral cobalt dose, vitamin B12 injection or intraruminal soluble glass bolus treatments also used. No growth responses were obtained and unsupplemented animals generally had an adequate cobalt and selenium status. No toxicity problems were encountered from either element.

Animals↗