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R C Davis

Publications and source records attributed to R C Davis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Lipase engineering: a window into structure-function relationships.

Utilization of genetic engineering techniques to create novel functional lipases has increased knowledge of structure-function relationships in this important class of enzymes. The examples of engineered lipases presented in this chapter addressed the investigation of domain-specific properties, heparin binding, and subunit orientation. Conclusions reached are credible because the designed lipases retained catalytic activity, implying native, or near-native, conformation. This approach has demonstrated vigor by determining the domain location of several important enzyme functions and by providing the first evidence that LPL subunits are arranged in a head-to-tail orientation. In conjunction with physical techniques, such as crystallography and nuclear magnetic resonance spectroscopy, the engineered lipase approach could reveal new insights into the mechanism by which lipolysis is accomplished. The studies described here represent only the first attempts to explore that subject; more sophisticated lipase engineering will be used in future as a window into structure-function relationships.

Amino Acid Sequence↗
PubMed1996

Increases in serum unbound free fatty acid levels following coronary angioplasty.

Serum unbound free fatty acid levels (FFAu) were measured in patients undergoing percutaneous transluminal coronary angioplasty (PTCA) using the fluorescent probe acrylodan intestinal fatty acid binding protein (ADIFAB). These are the first measurements of FFAu, under nonphysiologic conditions. In these studies, FFAu, levels were determined in 22 patients 5 minutes before and 30 minutes after the procedure. Post-PTCA FFAu, levels were higher than pre-PTCA levels in all patients. The average post-PTCA level for all patients was 103 nM, about 14-fold higher than the 7.5 nM value observed in healthy subjects. Although all patients exhibited elevated FFAu levels after PTCA, ischemic ST-segment changes were observed in only 11 of these patients. The average post-PTCA FFAu levels for patients with significant ST-segment changes (123 nM) were significantly higher than those in patients who did not exhibit such changes (47 nM). Average FFAu (22 nM) levels before the procedure were elevated in the patient population relative to healthy subjects and these values correlated positively with post-PTCA levels. These results suggest that increased serum FFAu levels reflect angioplasty-induced ischemia and that FFAu levels may provide a more sensitive measure of ischemia than electrocardiographic measurements. Moreover, because 30% of these patients had post-PTCA FFAu concentrations exceeding those found to alter in vitro cell function, the increased serum FFAu levels that accompany ischemia may be deleterious for myocardial function.

Adult↗
PubMed1996

Domain-structure analysis of recombinant rat hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.

Amino Acid Sequence↗
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↗
PubMed1996

Adjusting to criminal victimization: the correlates of postcrime distress.

This article explores the correlates of immediate and short-term psychological distress among victims of burglary, robbery, and nonsexual assault. A panel design was employed. Crime victims were interviewed within 1 month following the incident and again 3 months later. Four sets of predictors were examined: demographics, previctimization adjustment and stress, features of the crime incident, and victims' perceptions. Measures of distress included a range of standard indices of adjustment and symptomatology. Demographic characteristics and victim perceptions accounted for the greatest proportions of variance in the outcome measures at Time 1 and Time 2. The strongest predictors of psychological adjustment at the end of 3 months included adjustment after 1 month, education, victim injury, victims' beliefs that their lives had been endangered during the crime episode, and victims' appraisals of the world as meaningful. Implications for treatment and directions for future studies are discussed.

Adaptation, Psychological↗
PubMed1996

Translational regulation of lipoprotein lipase by thyroid hormone is via a cytoplasmic repressor that interacts with the 3' untranslated region.

To better characterize the increase in lipoprotein lipase (LPL) translation by hypothyroidism, adipocytes were prepared from control and hypothyroid rats. Whereas LPL synthesis was higher in hypothyroid adipocytes, with no change in mRNA levels, there was no increase in hormone-sensitive lipase (HSL) synthesis. To determine whether a transacting translation regulatory factor was present, a cytoplasmic fraction was prepared from control and hypothyroid adipocytes, and added to an in vitro translation system containing the hLPL mRNA. The hypothyroid cell fraction from adipose and heart yielded an increase in LPL translation, when compared to control extracts. Further experiments determined that the control adipocyte extract contained a translation-inhibitory factor that was 8-fold lower in activity in the hypothyroid extract. Using different LPL mRNA constructs in the in vitro translation reaction, the region that controlled translation was localized to nucleotides 1599 to 1638 (proximal 3' untranslated region (UTR)). To confirm the presence of a transacting factor, a sense RNA strand corresponding to this region was added to the in vitro translation reaction. This sense strand competed for the transacting factor in the control cell extract, yet had no effect on the hypothyroid cell extract. Thus, there is a translation repressor factor in the cytoplasm of rat adipocytes, and this factor is greatly reduced in activity in hypothyroid rat adipocytes. Because a similar mechanism of LPL regulation occurs in response to epinephrine, the absence of the translation repressor may be a mechanism for the loss of sensitivity of hypothyroid cells for catecholamines.

Adipose Tissue↗
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↗