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Biomedical subjects

D H Gregory

Publications and source records attributed to D H Gregory.

At least 19 recordsLinked to original sources
PubMed1993

Interaction of thymidylate synthase with pyridoxal 5'-phosphate as studied by UV/visible difference spectroscopy and molecular modeling.

Pyridoxal 5'-phosphate (PLP) is an effective inhibitor of Lactobacillus casei thymidylate synthase (TS), competitive with respect to the nucleotide substrate dUMP (Chen et al., 1989). The UV/vis difference spectra of TS-PLP complexes show lambda max at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, and lambda min at 388 nm due to depletion of free PLP. At high concentrations of PLP a new absorbance at 430 nm forms due to nonspecific Schiff base formation between PLP and lysine residues of the enzyme. Using spectral titration at 328 nm, the binding constant of the specific TS-PLP complex was determined to be 0.5 microM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP complex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules which bind to the active site of TS. Analogs of PLP which do not contain the phosphate or the aldehyde moieties of PLP bound poorly to the enzyme, thus demonstrating the importance of these functional groups for binding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as the wild-type enzyme. Treatment of the C198A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Prediction of fluorine chemical shifts in proteins.

Molecular dynamics calculations have been used in an effort to estimate the change in fluorine nmr shielding when a fluorine nucleus enters the tertiary structure of a protein. Considerations of the possible interactions that can define the shift parameter change suggest that van der Waals interactions are the leading determinant of fluorine shifts in proteins, although aromatic ring currents, other magnetic anisotropies, and electrostatic field effects could result in shift distinctions of 1 ppm or smaller. Results of our studies of a fluorine-containing analogue of the ribonuclease A S-protein/S-peptide complex indicate that static structures such as those implied by crystallographic data lead to overestimates of the magnitude of the van der Waals shielding term; molecular dynamics simulations provide indications of the effects of conformational averaging in defining this term. The treatment used predicts the correct direction of the shift change when the fluorine enters this protein environment from aqueous solution and, with an experimentally supported choice of adjustable parameters, gives agreement with the magnitude of the shift.

Anisotropy↗
PubMed1987

Fluorine nuclear magnetic resonance spectra of rabbit carbonmonoxyhemoglobin.

Carbonmonoxyhemoglobin prepared from protein isolated from rabbits maintained on a diet supplemented with 4-fluorophenylalanine (Phe (4F)) has been studied by fluorine NMR spectroscopy. Substitution of Phe(4F) appears to take place randomly at the sixteen nonequivalent phenylalanine positions of the globins; examination of hybrid hemoglobins in which only one type of globin chain contained the fluorinated amino acid, as well as changes in the spectrum upon exposure to oxygen, aided in the assignment of fluorine resonances from the alpha- or beta-globin chains. The effects of modification of Cys-beta-93 with a spin label and variation of pH and sample temperature on the spectrum were also examined. These data in association with theoretical estimates of aromatic ring current and van der Waals effects on chemical shifts were used to support tentative assignments of several signals observed to specific amino acid residues. Evidence suggesting the presence of two conformational forms of the protein, possibly due to disorder of the heme groups, is described.

Animals↗
PubMed1979

Acute viral hepatitis with bridging hepatic necrosis. An overview.

Bridging hepatic necrosis in the setting of acute viral hepatitis (BHN/AVH) represents an enigmatic syndrome inasmuch as its incidence, significance, course, and therapeutic response have not been clearly defined. It has been thought that this histologic finding carries a high risk of early mortality or evolution to chronic active hepatitis and/or cirrhosis. The data are sparse, and largely based on retrospective studies in selected populations. Steroids have not proved to be effective thus far, while drugs used in other forms of serious liver disease (eg, penicillamine, colchicine) have not been tried. A recent prospective study indicates that BHN/AVN may be a far more benign entity than was previously suspected. Further prospective studies are needed to clarify the significance of this lesion as well as the need for and response to medical therapy.

Adrenal Cortex Hormones↗
PubMed1978

Preferential utilization of free cholesterol from high-density lipoproteins for biliary cholesterol secretion in man.

High- and low-density lipoproteins carrying free cholesterol labeled with 3H or 14C were administered to a patient with a bile fistula. The free cholesterol from high-density lipoproteins was more rapidly incorporated into biliary cholesterol than the free cholesterol from low-density lipoproteins. These findings show that the liver in man selectively utilizes and secretes the free cholesterol from a particular lipoprotein.

Aged↗
PubMed1978

Multicompartmental analysis of cholesterol metabolism in man. Characterization of the hepatic bile acid and biliary cholesterol precursor sites.

The present report has presented the first clear evidence in man for the existence of specific hepatic cholesterol precursor sites associated with the formation and secretion of bile acids and biliary cholesterol. These hepatic compartments derive virtually all their cholesterol from newly synthesized and lipoprotein free cholesterol. The model which is presented was formulated on current concepts of cholesterol metabolism in man and is concerned, at this initial stage, with the elucidation of the bile acid and biliary cholesterol compartments. The complexity of cholesterol metabolism in man necessitated an initial approach that would minimize the number of inputs of cholesterol into the system, allow for the sampling of several cholesterol compartments, and permit the simultaneous labeling of newly synthesized cholesterol and preformed cholesterol. To achieve these objectives, we studied the patient with a total bile fistula. Six patients were administered simultaneously pulse injections of labeled mevalonic acid and [(14)C]cholesterol. The qualitative features of the specific activity time course curves after labeled mevalonic acid revealed no precursor-product relationship between bile acid, biliary cholesterol, and plasma free cholesterol. The peak specific activity of the bile acids was reached in approximately 100 min and was higher than the biliary cholesterol, which was higher than the plasma free cholesterol. The plasma free cholesterol specific activity became higher than the other lipids after 12 h and remained higher throughout the period of study. Similar related observations were made with [(14)C]cholesterol. The data were then subjected to simulation analysis and modeling using the SAAM-27 computer program. Computer least-square fits of the data were obtained after the model was evolved. During the model development, the least number of compartments and transport pathways were introduced consistent with a good fit of the data. Of particular importance was the constraint that the model fit the data obtained from both [(14)C]cholesterol and labeled mevalonic acid. The same parameter values were used to fit the data from both tracers. The fluxes arrived at in the model indicate that 31% and 20%, respectively, of the cholesterol input into the bile acid and biliary cholesterol precursor sites were derived directly from the newly synthesized hepatic cholesterol. The remainder had its origin predominantly from lipoprotein free cholesterol. Plasma esterified cholesterol (as free) made a small contribution (11%) to the bile acid compartment. Similarly, 10% of the biliary cholesterol arose from an unknown hepatic site. The present report has provided the basis for a new procedure for studying in vivo cholesterol metabolism in man. Examination of the derived cholesterol flux rates between the compartments suggests the presence of an important mechanism regulating the partitioning of lipoprotein free cholesterol between the bile acid and biliary cholesterol precursor sites. Aberrations in the proportioning of precursor cholesterol between these sites could be a causative factor precipitating the excessive secretion of biliary cholesterol and the production of lithogenic bile.

Aged↗
PubMed1976

Quantitative aspects of the conversion of 5 beta-cholestane intermediates to bile acids in man.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.

Bile Acids and Salts↗
PubMed1975

Mechanism of secretion of biliary lipids. I. Role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lecithin and cholesterol.

The role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lipids was investigated by using the isolated perfused rat liver model. Labeled lecithin precursors ((3H)-palmitic acid, (14C)linoleic acid, (3H)choline, and 32PO4) and a cholesterol precursor ((3H)mevalonic acid) were administered with and without sodium taurocholate. The incorporation pattern of these labeled precursors into linoleyl and arachidonyl lecithins and cholesterol fractions of microsomes, bile canaliculi, and bile were examined at 30-min intervals up to 90 min. Marker enzymes and electron microscopy indicated that isolated subfractions of plasma membranes were enriched with bile canaliculi (less than 10 percent microsomal contamination). Taurocholate significantly stimulated the incorporation of 32PO4, (3H)choline, (3H)palmitic acid, and (14C)linoleic acid into linoleyl and arachidonyl lecithin with parallel incorporation curves for microsomal and bile canalicular membranes throughout the 90-min study period. During the 30-60-min period, however, these same lecithin fractions in bile significantly exceeded the specific activity of the membrane lecithins. The enzyme CDP-choline diglyceride transferase was virtually absent from canaliculi relative to microsomes, indicating that canaliculi lack the capacity for de novo lecithin synthesis. Incorporation of (3H)mevalonic acid into membranous and biliary cholesterol followed a pattern similar to that for lecithin. These data provide evidence that (a) biliary lecithin and cholesterol are derived from a microsomal subpool regulated by the flux of enterohepatic bile acids, (b) the role of the bile canalicular membranes with respect to biliary lipids is primarily transport rather than synthesis, and (c) lecithin and cholesterol are transported together from microsomes to bile. The findings are consistent with the existence of a cytoplasmic lipid complex within the hepatocyte which is actively involved in the intermembrane transport of biliary lipid.

Animals↗
PubMed1975

Evidence for the existence of definitive hepatic cholesterol precursor compartments for bile acids and biliary cholesterol in man.

A bile fistula patient was administered intravenously a constant infusion of [3H]mevalonic acid for 4 hrs, and then after 2 weeks he was given a pulse of [3H]mevalonic acid. Bile and blood were collected at frequent intervals. The specific activity-time course curves did not show a precursor-product relationship between biliary cholesterol, plasma free cholesterol and bile acids. Both the constant infusion and pulse labeling data indicated that the bile acid precursor had a more rapid rate of turnover than plasma or biliary cholesterol; the biliary cholesterol precursor turned over more rapidly than plasma cholesterol. The data suggest the presence of multiple hepatic cholesterol precursor compartments. Bile acids may be derived predominantly from newly synthesized cholesterol in man.

Bile Acids and Salts↗