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Biomedical subjects

R C Davis

Publications and source records attributed to R C Davis.

At least 55 records · Page 3Linked to original sources
PubMed1994

Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme.

Lipoprotein lipase (LPL) and hepatic lipase (HL) share two conserved asparagine-linked glycosylation sites, located at the amino- and carboxy-terminal domains of the protein. Human HL contains two additional sites, preceding each conserved site by 36 and 35 amino acids, respectively. The utilization of these sites for glycan-binding and the role of each glycan chain for the catalytic function of human LPL, rat HL, and human HL was investigated. To accomplish this aim, potential Asn glycosylation sites were changed to Gln by site-directed mutagenesis and the resulting constructs were expressed in a mammalian (COS) cell system. We demonstrate the following. 1) All potential glycosylation sites in human LPL, rat HL, and human HL are utilized. 2) Lack of glycosylation at the two non-conserved sites in human HL has no effect on enzyme expression. 3) Glycosylation at the conserved Asn sites in the N-terminal domain of LPL and HL is required for the synthesis of a fully active and secreted lipase. While this is an absolute requirement for LPL, a portion (approximately 25%) of HL molecules lacking glycosylation at this essential site still becomes active and secreted. However, the simultaneous elimination of both glycosylation sites at the N-terminal domain of human HL results in the virtual abolishment of enzymatic activity and secretion. 4) Glycosylation at the conserved sites in the C-terminal domain is not essential for the expression of active lipases. 5) Eliminating all glycosylation sites in LPL and HL results in the synthesis of inactive enzymes that are retained intracellularly; however, a small portion (2%) of unglycosylated rat HL was active and secreted. We conclude that glycosylation overall plays an important role in the formation of functional LPL and HL.

Animals↗
PubMed1993

Use of angioplasty in the management of complicated perioperative infarction following bypass surgery.

Ischemic complications in the perioperative period following bypass surgery adversely affect both short- and long-term prognosis. Coronary angioplasty was successfully performed in 2 patients sustaining complicated postoperative myocardial infarctions with resolution of angina and restoration of hemodynamic stability. These cases illustrate that angioplasty can be done safely in such patients and may favorably alter short-term outcome.

Aged↗
PubMed1993

Chromosomal localization of lipolytic enzymes in the mouse: pancreatic lipase, colipase, hormone-sensitive lipase, hepatic lipase, and carboxyl ester lipase.

Several lipases and their cofactors are involved in the absorption, transport, storage, and mobilization of lipids. As part of an effort to examine the role of these enzymes in plasma lipid metabolism and genetic susceptibility to atherosclerosis, we report the chromosomal mapping of their genes in mouse. Restriction fragment length variants for each gene were identified, typed in an interspecific cross, and tested for linkage to known chromosomal markers. The gene for pancreatic lipase resides on chromosome 19, while the gene for its cofactor, colipase, is on chromosome 17. A gene for a protein with sequence similarity to pancreatic lipase was tightly linked (no observed recombination) to the gene for pancreatic lipase, suggesting a gene cluster. The gene for hormone-sensitive lipase is near the gene cluster containing apolipoproteins C-II and E on chromosome 7. The gene for hepatic lipase is near the gene for apolipoprotein A-I on chromosome 9. The carboxyl ester lipase gene resides on chromosome 2. Previously, we have mapped the gene for lipoprotein lipase to chromosome 8. Thus, with the exception of pancreatic lipase and a related protein, these lipase genes, including several that are members of a gene family, are widely dispersed in the genome. Comparison of chromosomal locations for these genes in mouse and humans shows that the previously observed interspecies syntenies are preserved.

Animals↗
PubMed1992

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.

Amino Acid Sequence↗
PubMed1992

Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.

The relationship between maturation of lipoprotein lipase (LPL) and its translocation from the endoplasmic reticulum (ER) to the Golgi complex was determined by measuring lipolytic activity under conditions preventing transport of the enzyme from the ER to the Golgi compartment. In the presence of brefeldin A, a reagent that inhibits movement of proteins from the ER and causes the disassembly of the Golgi complex, pro-5 Chinese hamster ovary cells accumulated catalytically active LPL, while secretion of the enzyme was effectively blocked. LPL retained intracellularly by brefeldin A treatment possessed oligosaccharide chains that were processed to the complex form by the Golgi enzymes redistributed into the ER. At 16 degrees C, a condition disrupting protein transport to the cis-Golgi, the retained enzyme again remained catalytically active although the oligosaccharides remained in the high mannose form. Lastly, attachment of the specific ER retention signal KDEL (Lys-Asp-Glu-Leu) to the carboxyl terminus of LPL also resulted in intracellularly retained enzyme that was fully active. The importance of oligosaccharide processing for attainment of LPL catalytic activity in vitro was also determined. LPL was active and secreted when trimming of the mannose residues was inhibited by deoxymannojirimycin and when addition of complex sugars was blocked using Chinese hamster ovary mutants (lec1 and lec2), indicating that these processing events are not necessary for the expression of a functional enzyme. However, blocking glucose removal by glucosidase inhibitors (castanospermine and N-methyl-deoxynojirimycin) resulted in a significant reduction in LPL specific activity and secretion. Thus, glucose trimming of LPL oligosaccharides is essential for enzyme activation; however, further oligosaccharide processing or translocation of the enzyme to the cis-Golgi is not required for full expression of lipolytic activity in vitro.

Amino Acid Sequence↗
PubMed1991

Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.

Hepatic lipase and lipoprotein lipase hydrolyze fatty acids from triacylglycerols and are critical in the metabolism of circulating lipoproteins. The two lipases are similar in size and amino acid sequence but are distinguished by functional differences in substrate preference and cofactor requirement. Presumably, these distinctions result from structural differences in functional domains. To begin localization of these domains, a chimeric lipase was constructed composed of the N-terminal 329 residues of rat hepatic lipase linked to the C-terminal 136 residues of human lipoprotein lipase. The chimera hydrolyzed both monodisperse short-chain (esterase) and emulsified long-chain (lipase) triacylglycerol substrates with catalytic and kinetic properties closely resembling those of native hepatic lipase. However, monoclonal antibodies to lipoprotein lipase inhibited the lipase activity, but not the esterase function, of the chimera. Therefore, the chimeric molecule is a functional lipase and contains elements and characteristics from both parental enzymes. It is proposed that the N-terminal domain, containing the active center from hepatic lipase, governs the catalytic character of the chimera, and the C-terminal domain is essential for hydrolysis of long-chain substrates.

Amino Acid Sequence↗
PubMed1991

Expression of biologically active hormone-sensitive lipase in mammalian (COS) cells.

cDNAs encoding rat adipose tissue hormone-sensitive lipase were expressed in COS cells, under the control of the SV40 promoter to half the level in rat adipocytes, the richest native source of the enzyme. A cDNA lacking most of the long 5'-untranslated region of the full-length rat hormone-sensitive lipase cDNA was, with regard to the lipase activity, on the average 70% more efficiently expressed that the full-length cDNA. The recombinant protein was almost identical to hormone-sensitive lipase of rat adipose tissue with respect to specific activity, susceptibility to inhibitors, molecular size, phosphorylation and activation by cyclic AMP-dependent protein kinase. The described eukaryotic expression system will allow analysis of effects of amino acid substitutions introduced into the lipase molecule by site-directed mutagenesis.

Adenosine Monophosphate↗
PubMed1991

Supportive and unsupportive responses of others to rape victims: effects on concurrent victim adjustment.

Evaluated the impact that actions of significant others have on adjustment following rape. Significant other behavior is conceptualized as having two dimensions--supportive behavior and unsupportive behavior--and each dimension was measured using multiple items. Unsupportive behavior, but not supportive behavior, was found to bear a significant association to victim adjustment. Implications for those who work with victims are discussed.

Adaptation, Psychological↗
PubMed1991

A two-cycle immunoprecipitation procedure for reducing nonspecific protein contamination.

A two-cycle immunoprecipitation procedure is described that markedly reduces nonspecific protein contamination occurring during the precipitation of hepatic lipase from rat H4 hepatoma cells. In this method, the precipitation of immune complexes during both cycles is achieved by utilizing a sodium dodecyl sulfate (SDS)-washed preparation of lyophilized Staphylococcus aureus cells (Staph A); this washed preparation effectively removes Staph A contaminants without compromising the ability to bind immune complexes. Following initial immunoprecipitation of the antigen, the Staph A/IgG/antigen complex containing coprecipitated nonspecific proteins was dissociated with SDS. Triton X-100 was added to the dissociated immunoprecipitate at a concentration (by weight) of at least 5 parts Triton X-100 to 1 part SDS. A second cycle of immunoprecipitation was then initiated by addition of fresh antibody, followed by Staph A precipitation of immune complexes and analysis by SDS-polyacrylamide gel electrophoresis. The two-cycle procedure is shown to be reproducible and suitable for the quantitative determination of relative amounts of hepatic lipase. The procedure described here is generally applicable to the immunoprecipitation of other antigens.

Antigen-Antibody Complex↗
PubMed1991

Assignment of human pancreatic lipase gene (PNLIP) to chromosome 10q24-q26.

Human pancreatic lipase (EC 3.1.1.3) is a 56-kDa protein secreted by the acinar pancreas and is essential for the hydrolysis and absorption of long-chain triglyceride fatty acids in the intestine. In vivo, the 12-kDa protein cofactor, colipase, is required to anchor lipase to the surface of lipid micelles, counteracting the destabilizing influence of bile salts. Southern blot analysis, using a pancreatic lipase cDNA to probe DNA from mouse-human somatic cell hybrids, indicated that the pancreatic lipase gene (PNLIP) resides on human chromosome 10. In situ hybridization to human metaphase chromosomes confirmed the cell hybrid results and further localized the gene to the 10q24-qter region with the strongest peak at q26.1.

Blotting, Southern↗
PubMed1991

Assignment of the human pancreatic colipase gene to chromosome 6p21.1 to pter.

Pancreatic colipase is a 12-kDa polypeptide cofactor for pancreatic lipase (EC 3.1.1.3), an enzyme essential for the absorption of dietary long-chain triglyceride fatty acids. Colipase is thought to anchor lipase noncovalently to the surface of lipid micelles, counteracting the destabilizing influence of intestinal bile salts. Using primers derived from the known amino acid sequence, we have used the polymerase chain reaction to produce a cDNA clone corresponding to the complete coding region of the human procolipase mRNA. Southern blot analysis of genomic DNA from a panel of mouse-human somatic cell hybrids indicated that the colipase gene (CLPS) resides on human chromosome 6. Further analysis of somatic cell hybrids carrying chromosome 6 translocations permitted regional localization of CLPS to the 6p21.1-pter region.

Blotting, Southern↗
PubMed1991

Extracorporeal shock wave lithotripsy of gallstones: results and 6-month follow-up in 141 patients.

A study of biliary shock wave lithotripsy of gallstones sponsored by Dornier Medical Systems, Munich, began in the United States in May 1988 to evaluate the efficacy and safety of extracorporeal shock wave lithotripsy (ESWL) and the need for adjunctive therapy with ursodeoxycholic acid (UDCA). One hundred forty-one symptomatic patients with one to three gallstones 5-30 mm in diameter were randomized to treatment. One week before ESWL, patients were given either UDCA or placebo. This treatment was continued for 6 months. All patients underwent follow-up at predetermined intervals. According to the protocol, re-treatment for fragments larger than 5 mm in diameter could be performed only at 6 weeks; 26 (18%) of the 141 patients were retreated. At 6 months, the stone-free rates for single stones were as follows: patients with noncalcified stones receiving UDCA, 29%; patients with noncalcified stones receiving placebo, 24%; and patients with partially calcified stones receiving either UDCA or placebo, 6%. No significant difference was noted between the UDCA and placebo groups. At 6 months, the stone-free rates in patients with single, noncalcified stones 20 mm or less in diameter were 40% (UDCA) and 32% (placebo), which is superior to rates for those with solitary, noncalcified gallstones 21-30 mm in diameter and those with two or three stones.

Adult↗
PubMed1991

Familial chylomicronemia (type I hyperlipoproteinemia) due to a single missense mutation in the lipoprotein lipase gene.

Complete deficiency of lipoprotein lipase (LPL) causes the chylomicronemia syndrome. To understand the molecular basis of LPL deficiency, two siblings with drastically reduced postheparin plasma lipolytic activities were selected for analysis of their LPL gene. We used the polymerase chain reaction to examine the nine coding LPL exons in the two affected siblings and three relatives. DNA sequence analysis revealed a single nucleotide change compared with the normal LPL cDNA: a G----A substitution at nucleotide position 680. This transition caused a replacement of glutamic acid for glycine at amino acid residue 142 of the mature LPL protein. Amino acid sequence comparisons of the region surrounding glycine-142 indicated that it is highly conserved among lipases from different species, suggesting a crucial role of this domain for the LPL structure. Expression studies of the mutant LPL cDNA in COS-7 cells produced normal amounts of enzyme mass. However, the mutated LPL was not catalytically active, nor was it efficiently secreted from the cells. This established that the Gly----Glu substitution at amino acid 142 is sufficient to abolish enzymatic activity and to result in the chylomicronemia syndrome observed in these patients.

Amino Acid Sequence↗