PubMed Health⌕ Search

Biomedical subjects

R C Davis

Publications and source records attributed to R C Davis.

At least 19 recordsLinked to original sources
PubMed1996

Human hepatic lipase subunit structure determination.

Chinese hamster ovary cells were stably transfected with a human hepatic lipase (HL) cDNA. The recombinant enzyme was purified from culture medium in milligram quantities and shown to have a molecular weight, specific activity, and heparin affinity equivalent to HL present in human post-heparin plasma. The techniques of intensity light scattering, sedimentation equilibrium, and radiation inactivation were employed to assess the subunit structure of HL. For intensity light scattering, purified enzyme was subjected to size exclusion chromatography coupled to three detectors in series: an ultraviolet absorbance monitor, a differential refractometer, and a light scattering photometer. The polypeptide molecular weight (without carbohydrate contributions) was calculated using the measurements from the three detectors combined with the extinction coefficient of human HL. A single protein peak containing HL activity was identified and calculated to have a molecular mass of 107,000 in excellent agreement with the expected value for a dimer of HL (106.8 kDa). In addition, sedimentation equilibrium studies revealed that HL had a molecular mass (with carbohydrate contributions) of 121 kDa. Finally, to determine the smallest structural unit required for lipolytic activity, HL was subjected to radiation inactivation. Purified HL was exposed to various doses of high energy electrons at -135 degrees C; lipase activity decreased as a single exponential function of the radiation dose to less than 0.01% remaining activity. The target size of functional HL was calculated to be 109 kDa, whereas the size of the structural unit was determined to be 63 kDa. These data indicate that two HL monomer subunits are required for lipolytic activity, consistent with an HL homodimer. A model for active dimeric hepatic lipase is presented with implications for physiological function.

Animals↗
PubMed1996

Physician practices regarding anticoagulation and cardioversion of atrial fibrillation.

BACKGROUND: Stroke is one of the most significant potential complications in patients who are undergoing cardioversion for atrial fibrillation. To minimize the risk of stroke, the American College of Chest Physicians' (ACCP's) Third Consensus Conference on Antithrombotic Therapy developed specific recommendations regarding anticoagulation before and following elective cardioversion of patients with atrial fibrillation. OBJECTIVE: To determine if patients undergoing cardioversion for atrial fibrillation are administered anticoagulants according to the ACCP's Third Consensus Conference on Antithrombotic Therapy recommendations. DESIGN: A retrospective review of cases of atrial fibrillation at a tertiary care teaching hospital to determine if physicians are routinely following these recommendations. METHODS: Data were collected for the year 1994 for all patients admitted to a tertiary care teaching hospital with a diagnosis of atrial fibrillation (n = 111). The ACCP's recommendations that were evaluated included the following: patients undergoing elective cardioversion for atrial fibrillation should receive anticoagulation for 3 weeks before and 4 weeks following cardioversion except in cases of new-onset atrial fibrillation, and warfarin and heparin should be administered jointly for several days before discontinuation of heparin therapy. RESULTS: Of the 111 patients who presented with a diagnosis of atrial fibrillation, 51 underwent elective cardioversion. In 18 (35%) of 51 cases, physicians failed to follow at least one of ACCP's recommendations regarding anticoagulation. These included failing to (1) administer anticoagulants to patients for 3 weeks before elective cardioversion (n = 14); (2) administer anticoagulants to patients for 4 weeks following cardioversion (n = 6); and (3) overlap heparin and/or warfarin therapies for 72 hours (n = 4). Six cases failed to meet more than one of these recommendations. CONCLUSION: Physicians are not routinely following the ACCP's Third Consensus Conference on Antithrombotic Therapy recommendations regarding anticoagulation in elective cardioversion of atrial fibrillation, thus increasing patients' risk of stroke.

Adult↗
PubMed1995

Clinical and angiographic correlates of normal creatine kinase with increased MB isoenzymes in possible acute myocardial infarction.

A retrospective study of patients with possible acute myocardial infarction was conducted over a 2-year period to evaluate the clinical characteristics, angiographic findings, and in-hospital prognosis in patients with normal total creatine kinase (CK) activity and increased MB isoenzyme activity (CK-MB). Thirty-nine cases were identified (study group) and compared with cases of Q-wave (n = 77) and non-Q-wave (n = 60) infarctions. Compared with the Q-wave group, study group patients were older (67.5 +/- 9.0 vs 60.8 +/- 11.5 years; p < 0.01) and more often had previous diagnoses of coronary disease (52.6% vs 18.2%; p < 0.01) and peripheral vascular disease (28.9% vs 10.4%; p = 0.02). Angina (92.2% vs 65.8%; p < 0.01) and ST elevation (81.8% vs 13.2%; p < 0.01) were more common in the Q-wave group. Nearly identical clinical profiles and electrocardiographic findings were observed in the study and non-Q-wave groups. Angiographic analysis revealed a higher frequency of multivessel disease in the study group (89.6%) than in the Q-wave group (48.6%, p < 0.01) but no difference between the study group and the non-Q-wave group (79.6%; p not statistically significant). Left ventricular function and in-hospital complications were similar among groups. It is concluded that patients with normal total CK activity and increased CK-MB concentration represent a subgroup of patients with non-Q-wave infarction with a high prevalence of multivessel coronary disease.

Aged↗
PubMed1995

Regulation of lipoprotein lipase translation by epinephrine in 3T3-L1 cells. Importance of the 3' untranslated region.

Lipoprotein lipase (LPL) is a central enzyme in lipoprotein metabolism and is in part responsible for adipocyte lipid accumulation. Catecholamines are known to decrease the activity of LPL in adipocytes, and we have previously demonstrated that this inhibition occurs posttranscriptionally, with a prominent inhibition of LPL translation. To better characterize the inhibition of LPL translation, 3T3-L1 cells were differentiated into adipocytes, and exposed to epinephrine. Epinephrine induced a dose-dependent decrease in LPL synthesis using [35S]methionine incorporation, with no change in LPL mRNA levels, demonstrating translational regulation of LPL in this cell line. The poly A-enriched RNA from epinephrine-treated cells was translated well in vitro, and there was no difference in the polysome profiles from control and epinephrine-treated cells, suggesting that epinephrine did not affect mRNA editing, and did not induce an inhibition of translation initiation. To obtain evidence for the presence of an inhibitory factor, a cytoplasmic extract from control, and epinephrine-treated adipocytes was human. When compared to the control cell extract, the epinephrine-treated cell extract sharply inhibited LPL translation in vitro, yet had no effect on the translation of other mRNAs. Epinephrine-treated cells had fourfold more of this inhibitor activity than control cells, and this translation inhibition was partially reversed by heat treatment. To determine what region of the LPL mRNA was involved in the translation inhibition, different LPL constructs were synthesized. The inhibitory effect of the epinephrine-treated cell extract was dependent on the presence of the first 40 nucleotides of the 3' (untranslated region UTR) (nucleotides 1599-1638), whereas deletion of the 5' UTR and other areas of the 3' UTR had no effect on translation inhibition. When a sense RNA strand corresponding to this region was added to the in vitro translation reaction, it restored translation towards normal, suggesting that the sense strand was competing for a transacting binding protein. Thus, epinephrine-treated adipocytes produced a transacting factor, probably a protein, that interacted with a region on the LPL mRNA between nucleotides 1599 and 1638, resulting in an inhibition of translation. These studies add new insight into the hormonal regulation of LPL.

3T3 Cells↗
PubMed1994

Identification of the active site serine of hormone-sensitive lipase by site-directed mutagenesis.

The consensus pentapeptide GXSXG is found in virtually all lipases/esterases and generally contains the active site serine. The primary sequence of hormone-sensitive lipase contains a single copy of this pentapeptide, surrounding Ser-423. We have analyzed the catalytic role of Ser-423 by site-directed mutagenesis and expression of the mutant hormone-sensitive lipase in COS cells. Substitution of Ser-423 by several different amino acids resulted in the complete abolition of both lipase and esterase activity, whereas mutation of other conserved serine residues had no effect on the catalytic activity. These results strongly suggest that Ser-423 is the active site serine of hormone-sensitive lipase.

Amino Acid Sequence↗
PubMed1994

Lipoprotein lipase domain function.

Human lipoprotein lipase (LPL) monomer consists of two domains, a larger NH2-terminal domain that contains catalytic residues and a smaller COOH-terminal domain that modulates substrate specificity and is a major determinant of heparin binding. Analyses of NH2-terminal domain function were performed after site-directed mutagenesis of the putative active-site serine residue, while COOH-terminal domain function was assessed following reaction with a monoclonal antibody. The native enzyme and mutant LPL in which serine 132 was replaced with alanine, cysteine, or glycine were transiently expressed in COS-7 cells. Mutant proteins were synthesized and secreted at levels comparable to native LPL; however, none of the mutants retained enzymatic activity. The mutant with alanine replacing serine 132 was purified and shown to be inactive with both esterase and lipase substrates; however, binding to a 1,2-didodecanoyl-sn-glycero-3-phosphatidylcholine monolayer was comparable to native LPL. These results are consistent with a catalytic, and not a lipid binding, role for serine 132. To investigate the function of the smaller COOH-terminal domain, LPL lipolytic and esterolytic activities as well as heparin binding properties were determined after reaction with a monoclonal antibody specific for this domain. Lipolytic activity was inhibited by the monoclonal antibody, whereas esterolytic activity was only marginally affected, indicating that the LPL COOH-terminal domain is required for lipolysis, perhaps by promoting interaction with insoluble substrates. Also, the affinity of antibody-reacted LPL for heparin was not significantly different from that of LPL alone, suggesting that (i) the heparin-binding site is physically distinct from the COOH-terminal domain region required for lipolysis and (ii) binding of antibody did not cause dimer dissociation. A model is proposed for the two LPL domains fulfilling different roles in the lipolytic process.

Animals↗
PubMed1994

Hormone-sensitive lipase: structure, function, evolution and overproduction in insect cells using the baculovirus expression system.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in the hydrolysis of stored triacylglycerols and is thereby a key enzyme in lipid metabolism and overall energy homeostasis. The gene organization of human HSL indicates that each putative functional region is encoded by a different exon, raising the possibility that HSL is a mosaic protein. The catalytic serine (Ser423), as shown by site-directed mutagenesis, is encoded by exon 6. The phosphorylation site for cAMP-mediated activity control and a second site, which is presumably phosphorylated by 5' AMP-activated kinase, are encoded by exon 8, and a putative lipid-binding region is encoded by the ninth and last exon. Besides the catalytic site serine motif (GXSXG), found in virtually all lipases, a sequence similarity between the region surrounding the catalytic site of HSL and that of five prokaryotic enzymes has been found, but the functional basis of this is not yet understood. To resolve the 3-D structure of HSL, an expression system utilizing recombinant baculovirus and insect cells has been established. The expressed protein, 80 mg/l culture, has been purified to homogeneity and a partial characterization indicates that it has the same properties as HSL purified from rat adipose tissue.

Animals↗
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↗