Biomedical subjects

N Fidge

Publications and source records attributed to N Fidge.

At least 37 records · Page 2Linked to original sources

PubMed1993

The effect of the HMG-CoA reductase inhibitor simvastatin and of cholestyramine on hepatic apolipoprotein mRNA levels in the rat.

We have administered the hypolipaemic drugs simvastatin and cholestyramine to rats, both separately and together, to determine whether drug-induced changes in plasma apo lipoprotein levels occur through regulation of hepatic genes coding for apolipoproteins (apos) AI, AIV, E and B. Cholestyramine alone had little effect on either the plasma levels of the apolipoproteins studied or their hepatic mRNA levels. However, simvastatin, either alone or in combination with cholestyramine, produced marked changes in both of these parameters. While simvastatin plus cholestyramine lowered mRNAs for apo AIV, apo E and apo B by 50%, 37%, and 55%, respectively, the treatment resulted in an increase of 150% in apo AI mRNA. This combined drug treatment caused a lowering in plasma concentration of all four apolipoproteins which failed to reach significance with apo E. Relative to controls, the values decreased to 83 +/- 14% for apo AI, 67 +/- 8% for apo AIV and 73 +/- 15 for apo B. An important novel finding of the present study, was that hypolipaemic drugs caused the apo B mRNA levels in rat liver to decrease. Previous reports have shown that although dietary fat or hormones caused marked changes in the concentration of circulating lipoproteins, they failed to alter apo B mRNA content. We suggest that the synthesis of apo B is subject to both pre- and post-translational regulation.

Animals↗

PubMed1991

Application of reversed-phase high-performance liquid chromatography to the separation of apolipoproteins A-IV, A-I and E from rat high-density lipoprotein.

Apolipoproteins A-IV, A-I and E from rat high-density lipoprotein (HDL) were successfully purified by reversed-phase high-performance liquid chromatography (RP-HPLC), using a method which we have previously developed for the separation of apolipoproteins A-IV, A-I and E from human lymph chylomicrons [T. Tetaz, E. Kecorius, B. Grego and N. Fidge, J. Chromatogr., 511 (1990) 147]. Since analytical-scale RP-HPLC indicated that the C apolipoproteins from rat HDL coeluted with both apo A-IV and apo A-I, delipidated rat HDL was first subjected to preparative-scale size-exclusion HPLC (HPSEC) on a Serva Si300 column, which effectively separated the C apolipoproteins from all but apolipoprotein E. Fractions from HPSEC which were enriched for apolipoproteins A-IV, A-I or E were directly applied to RP-HPLC on a TSK Phenyl-5PW column. This procedure yielded fractions containing apolipoproteins A-IV, A-I or E which were pure as assessed by N-terminal sequencing and silver staining of sodium dodecyl sulphate-polyacrylamide gels.

Animals↗

PubMed1991

Effect of lovastatin on the interaction between high density lipoprotein and cultured rat adrenocortical cells.

Preincubation of rat adrenocortical cells with lovastatin inhibits high density lipoprotein (HDL3) interaction with rat adrenocortical cells in a dose- and time-dependent fashion. Lovastatin causes a decrease in the number of binding sites and a moderate increase in the affinity of HDL binding to cells. Lovastatin also produced a dose- and time-dependent inhibition of cholesterol synthesis in these cells. Dose-dependence, but not time-dependence, of the inhibition of cholesterol synthesis, correlates with that of HDL binding (r = 0.91, P less than 0.004). Incubation of cells with lovastatin for up to 24 h does not change their free or total cholesterol content. The inhibitory effect was apparently not due to a modification of HDL by lovastatin. These results indicate that lovastatin causes a down-regulation of HDL binding sites on cultured rat adrenocortical cells.

Adrenal Cortex↗

PubMed1991

Characterization of the discoidal complexes formed between apoA-I-CNBr fragments and phosphatidylcholine.

The structure, composition, and physico-chemical properties of lipid-protein complexes generated between dimyristoylphosphatidylcholine (DPMC) and the CNBr fragments of human apoA-I were studied. The fragments were separated by high performance liquid chromatography and purified on a reversed-phase column. The complexes with DMPC were isolated on a Superose column; their dimensions were obtained by gradient gel electrophoresis and by electron microscopy. The secondary structure of the protein in the complexes was studied both by circular dichroism and by attenuated total reflection infrared spectroscopy. The fragments 1 and 4 of apoA-I, containing, respectively, two and three amphipathic helices, recombined with the phospholipid to generate discoidal particles with sizes similar to that of apoA-I- and apoA-II-DMPC complexes. The infrared measurements indicated that in all complexes the apolipoprotein helical segments were oriented parallel to the phospholipid acyl chains and that the protein was located around the edges of the discs. Computer modelling of the complexes based on energy minimization techniques proposed a model for these particles in agreement with the dimensions measured experimentally. In conclusion, we propose that apoA-I and its longest CNBr fragments are able to generate discoidal particles with DMPC, with apolipoprotein helical segments oriented parallel to the acyl chains of the phospholipids.

Apolipoprotein A-I↗

PubMed1990

Separation of human apolipoproteins A-IV, A-I and E by reversed-phase high-performance liquid chromatography on a TSK Phenyl-5PW column.

A method has been developed for the rapid separation of the medium-molecular-weight apolipoproteins A-IV, A-I and E by high-performance liquid chromatography. Separations were achieved using a commercially available column of very low hydrophobicity (TSK Phenyl-5PW) in the reversed-phase mode rather than the conventional mode of hydrophobic interaction. Delipidated apolipoproteins were dissolved in 20 mM orthophosphoric acid (pH 2.3), applied to the column which was pre-equilibrated with the same buffer, and eluted with an increasing gradient of acetonitrile. Purified apolipoproteins were identified by a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, amino acid analysis and N-terminal sequence analysis. In one step the method can be used to separate the major human chylomicron apolipoproteins A-IV, A-I and E, following preliminary removal of apolipoprotein A-II and the C apolipoproteins by size-exclusion chromatography.

Amino Acid Sequence↗

PubMed1989

Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver.

The liver plays a major role in the metabolism of plasma high-density lipoprotein (HDL). Several groups have postulated, but others refuted, the existence of a classical membrane receptor which recognizes HDL. In the present study, we identified and purified two liver HDL-binding proteins of 120 kDa (HB1) and 100 kDa (HB2), with apparent specificity for HDL3 devoid of E apolipoprotein. The plasma membrane was the richest source of the HDL-binding protein. Both proteins bound A-I and A-II apolipoproteins and retained HDL-binding activity after final purification. HB1 activity, but not that of HB2, was lost after treatment with beta-mercaptoethanol, but reduction did not change the apparent molecular mass of either protein. Antibodies against HB1 or HB2 did not cross-react, and preliminary structural investigations provide evidence to suggest that HB1 and HB2 are not structurally related. We thus provide evidence for at least two liver plasma-membrane proteins which bind HDL apolipoproteins, suggesting that protein-protein interaction participates to some degree in the mechanism of HDL recognition by cells.

Animals↗

PubMed1989

Monoclonal antibodies to human A-I apolipoprotein and characterisation of cyanogen bromide fragments of apoA-I.

Several monoclonal antibodies to human A-I apolipoprotein were produced after immunising mice with pure delipidated apoA-I. These monoclonal antibodies were characterised for their ability to react with whole lipoproteins, apolipoproteins and fragments of apoA-I generated by cleavage with cyanogen bromide. The data suggest that production of monoclonal antibodies using apoA-I as antigen was influenced by two major epitopes subsequently localised to cyanogen bromide fragments 1 and 3, and have been designated antibodies 1----5 A-IB and 6----10 A-IB, respectively. Cyanogen bromide fragments were first purified to homogeneity before screening by competitive displacement or immunoblotting procedures. Definitive characterisation of one antibody series (1----5 A-IB) depended ultimately on Western blotting following isoelectric focusing of purified apoA-I fragments. This technique identified the epitope for these antibodies to fragment 1, an identification not fully concluded from competitive displacement studies. These studies have also revealed the presence of microheterogeneity in fragment 1 (as well as in fragment 4) of apoA-I, suggesting that structural variations in several regions may account for the polymorphism observed in this apolipoprotein.

Animals↗

PubMed1989

Quantitation of apolipoprotein A-IV in human plasma using a competitive enzyme-linked immunosorbent assay.

A method for measuring human apolipoprotein A-IV has been developed using the competitive enzyme-linked immunosorbent assay (ELISA) system. The assay described is relatively easy, rapid, and inexpensive to perform, uses convenient dilutions of plasma (1/8-1/32) but is sensitive enough to quantitate the apoA-IV content of lipoproteins following gel filtration of small (0.3-0.5 ml) volumes of plasma. The working range is 100-600 ng of apoA-IV per 50-microliters sample and the intra- and interassay coefficients of variations are 7.5 and 10.2% (means), respectively. The mean apoA-IV concentration of 100 subjects was found to be 16.4 +/- 5.4 mg/dl. The assay can be performed on untreated plasma samples which may be stored frozen (-20 degrees C) for up to 2 months.

Apolipoprotein A-I↗

PubMed1987

Effect of acute inflammation on rat apolipoprotein mRNA levels.

Hybridization studies using specific cDNA have been used to determine the mRNA levels for rat apolipoproteins AI, AII, AIV, and E in extracts of rat liver and intestine. The ratios of intestinal mRNA/liver mRNA for apolipoprotein AI (apo AI), apo AIV, and apo E were 1.3, 1.7, and 0.1, respectively. Apo AII mRNA was detected in the liver but not in the intestine. The mRNA levels for apo AII and apo AIV in rat liver decreased during inflammation to minimums of 40% and 25% of normal, respectively. The mRNA levels for apo AIV in the intestine, apo E in the liver and for apo AI in both the liver and intestine did not change significantly during inflammation. The time course for the decrease in the hepatic mRNA levels for apo AIV was similar to those previously observed for the negative acute-phase proteins albumin and transthyretin. The serum levels for apo AIV were not affected by inflammation.

Animals↗

PubMed1987

Effects of dietary cholesterol and hypothyroidism on rat apolipoprotein mRNA metabolism.

The effects of dietary cholesterol and hypothyroidism on the mRNA levels of rat apolipoproteins A-I, A-IV, and E were measured in extracts of rat liver and rat intestine by hybridization to specific cDNA. Four groups, each comprised of six rats, were fed diets consisting of normal laboratory rat chow and either no supplements (control); 5% lard, 1% cholesterol, and 0.3% taurocholic acid (CF); 5% lard, 1% cholesterol, 0.3% taurocholic acid, and 0.1% propylthiouracil (CF-PTU); or 0.1% propylthiouracil (PTU) for 32 days. At the conclusion of the diets, serum cholesterol, triiodothyronine, and thyroxine levels were measured. The average serum cholesterol concentrations for the four groups were 50.4 +/- 3.7, 75.6 +/- 15.3, 135.3 +/- 41.5, and 73.3 +/- 16.4 mg/dl, respectively. The presence of propylthiouracil in the diets significantly lowered triiodothyronine and thyroxine levels in the serum. The mRNA levels for apolipoproteins A-I and A-IV in rat liver decreased significantly after the feeding of the CF-PTU diet (31 +/- 4% and 32 +/- 3% of normal, respectively) and the PTU diet (34 +/- 8% and 43 +/- 12% of normal, respectively), but showed little change after the CF diet (88 +/- 16% and 108 +/- 15% of normal, respectively). The effects of dietary propylthiouracil on the hepatic mRNA levels for apolipoproteins A-I and A-IV imply a role for thyroid hormones in regulating the mRNA levels for these apolipoproteins in rat liver. ApoE mRNA levels in the rat liver decreased slightly after the CF-PTU diet (74 +/- 12% of normal) and after the PTU diet (73 +/- 10% of normal).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Amino acid sequence of rat apolipoprotein A-II deduced from the nucleotide sequence of cloned cDNA.

A rat apolipoprotein A-II cDNA clone was isolated from a rat liver cDNA library by in situ hybridization of bacteriophage plaques using a 32P-labeled human apoA-II cDNA as a probe. The cDNA insert from this clone was characterized by DNA sequencing. The amino acid composition derived from the DNA sequence data matched well with that of rat apoA-II reported earlier (Herbert et al. 1974. J. Biol Chem. 249: 5718-5724), indicating that the cDNA insert coded for rat apoA-II. Further evidence was provided by a comparison of the amino acid sequence of rat apoA-II obtained here with that of human apoA-II (Brewer et al. 1972. Proc. Natl. Acad. Sci. USA. 69: 1304-1308). While the rat apoA-II cDNA insert did not code for the entire presegment, it had the same COOH-terminal residues of the presegment as well as the same prosegment (Ala-Leu-Val-Arg-Arg) as in human preproapoA-II, suggesting that rat apoA-II was also synthesized initially as preproapoA-II. Mature rat apoA-II contains 79 amino acids. Residue 6 of mature rat apoA-II is Asp, while it is Cys in human apoA-II, and this would account for the absence of dimeric forms of rat apoA-II in plasma. While the overall amino acid sequence homology between rat and human apoA-II is about 50%, the amphipathic alpha-helical structures, which are responsible for lipid-binding, seem to be conserved in the two proteins. The size of rat apoA-II mRNA was estimated to be about 600 nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1985

Identification of a high density lipoprotein binding protein from adrenocortical membranes.

A protein band with an apparent molecular weight of 78,000 daltons has been identified in the solubilised plasma membrane extract of sheep adrenal cortex which binds HDL3 devoid of E apolipoprotein. Following 'Western' blotting, and development of the nitrocellulose strips with appropriate antisera and color reagent, the same band, unlike other cortical membrane proteins or albumin, bound AI and AII apolipoproteins. Human LDL bound weakly to the same band but more strongly to another two proteins of higher molecular weight. These studies confirm the same degree of specificity of HDL3 binding found with cultured adrenal cells and strengthen the suggested existence of a specific HDL receptor.

Adrenal Cortex↗

PubMed1984

Characteristics of the binding of high-density lipoprotein3 by intact cells and membrane preparations of rat intestinal mucosa.

We have investigated the binding of high-density lipoprotein (HDL3, d = 1.12-1.21 g/ml), and apolipoprotein E-deficient human and rat HDL, obtained by heparin-Sepharose affinity chromatography, to intact cells and membrane preparations of rat intestinal mucosal cells. Binding of 125I-labeled HDL3 to the basolateral plasma membranes was characterised by a saturable, specific process (Kd = 21 micrograms of HDL3 protein/ml, Bmax = 660 ng HDL3 protein/mg membrane protein) and E-deficient human HDL demonstrated a similar affinity for the binding site. The basolateral plasma membranes isolated from proximal and distal portion of rat small intestine showed similar binding affinities for HDL3, whereas the interaction of HDL with brush-border membranes was characterised by mainly nonspecific and nonsaturable binding. The binding of 125I-labeled HDL3 to basolateral plasma membranes was competitively inhibited by unlabeled HDL3 but less efficiently by unlabeled human LDL. The putative HDL receptor was not dependent on the presence of divalent cations but was markedly influenced by temperature and sensitive to pronase treatment. We have also demonstrated, using whole intestinal mucosal cells, that lysine and arginine-modified HDL3 inhibited binding of normal 125I-labeled HDL3 to the same extent as normal excess HDL3. These data suggest that basolateral plasma membranes of rat intestinal mucosal cells possess a specific receptor for HDL3 which contains mainly apolipoprotein A-I and A-II, and the mechanisms of recognition of HDL3 differ from those involved in binding to the B/E receptor.

Animals↗

PubMed1984

The hormonal stimulation of high-density lipoprotein binding, internalization and degradation by cultured rat adrenal cortical cells.

Several studies have shown that high-density lipoprotein stimulates steroidogenesis in rat tissues which have been treated with pituitary hormones. To determine whether these hormones can directly affect receptors for high-density lipoprotein, we have incubated cultured rat adrenal cortical cells with 125I-labeled human HDL3 and studied the effect of corticotrophin on the binding, internalization and degradation of this lipoprotein. ACTH stimulated all these parameters of HDL metabolism in a dose-dependent manner with maximal stimulation occurring between 10 and 20 mU/ml. The effect was temperature-dependent and showed target cell specificity. Although the hormone stimulated binding and internalization 5-6-fold, degradation of HDL3 was substantially less (2-fold) than anticipated. This suggests that the lipoprotein was taken up by vesicles resembling receptosomes which escape fusion with lysosomes; thus degradation of entrapped particles does not occur, while transfer of cholesterol for steroidogenesis is unaffected.

Adrenal Cortex↗

PubMed1984

Synthesis of rat apolipoprotein E by Escherichia coli infected with recombinant bacteriophage.

A cDNA library was constructed from rat liver polyadenylated RNA using the expression vector lambda gt11-Amp3. Several clones expressing antigenic determinants for rat apolipoprotein E were identified. The cDNA insert in one clone was further characterized and found to have a sufficient length (1120 base pairs) to code for full length apolipoprotein E. Restriction mapping and nucleotide sequencing showed the clone to contain the coding region for apolipoprotein E flanked by about 120 nucleotides at the 3'-side and by about 64 nucleotides on the 5'-side. One of the proteins produced by the clone was found to be a prokaryotic/eukaryotic hybrid protein reacting with antibodies to both bacterial beta-galactosidase and rat apolipoprotein E.

Animals↗

PubMed1983

Distribution of C apolipoproteins between the vascular and lymph compartments of the rat.

This study has investigated the kinetics of transfer of C apolipoproteins between the vascular and lymph compartments of the rat. Very-low-density lipoprotein, labeled with [125I]apolipoprotein C, was injected intravenously into lymph duct-cannulated rats and the redistribution of radioactivity between lymph and plasma followed at frequent intervals for 3 h. Equilibration between the two compartments was rapid (10-15 min), and thereafter removal from both compartments continued at similar rates. Specific radioactivity determinations showed that lymph C-III-0, C-III-3, and C-III-2,1 apolipoproteins rapidly reached values identical to those of corresponding plasma C apolipoproteins and the interrelationship between the curves were consistent with precursor-product relationships in which all, or most, of the product (lymph apolipoprotein C-III) was derived from the precursor (plasma). However, the specific radioactivity curves for C-II peptide did not cross; the lower value for lymph C-II apolipoprotein suggests that, unlike C-III apolipoproteins, a substantial proportion (approx. 40%) of lymph C-II peptide is not derived from the plasma compartment. The most likely source of the unlabeled lymph apolipoprotein C-II is synthesis and secretion from the intestine.

Animals↗

PubMed1983

HDL3 degradation by proteases in isolated rat intestinal mucosal cells.

Human 125I-labelled HDL3 is degraded by isolated rat intestinal mucosal cells. In our experimental conditions, lipoprotein degradation occurred by two different mechanisms. In one, lipoprotein was degraded within the cell, following binding and internalisation. In the other, degradation occurred in the medium, which seemed to contain protease activity released from cells during incubation. Though lipoprotein-deficient serum apparently interfered with degradation in the medium, bovine serum albumin had no such effect. The lysosomal inhibitor, chloroquine, reduced degradation by 60% without inhibiting HDL binding. Intestinal cell extracts contained at least two different proteases, with pH optima of 4.5 and 8.0, respectively. Comparing HDL and LDL degradation on a molar basis, more HDL particles were degraded by the cell-free extracts at pH 4.5. This degradation was activated by dithiothreitol and was inhibited by iodoacetic acid. From these observations we conclude that HDL3 is taken up by the rat intestinal mucosal cell through a specific binding site and subsequently degraded by a thiol-dependent protease in the lysosome.

Animals↗

PubMed1983

Heterogeneity of very-low-density lipoprotein metabolism in hyperlipidemic subjects.

Very-low-density lipoproteins (VLDL) are triglyceride-rich lipoproteins that have been shown, by physicochemical means, to comprise more than one group of particles. Because of the potential atherogenicity of catabolized VLDL, we used the technique of heparin-affinity chromatography to separate VLDL into two classes of particles, one of which appears to contain partly catabolized VLDL. This observation is based on the higher cholesterol/triglyceride and higher apoprotein E/apoprotein C ratios in VLDL particles that are bound to heparin, resembling in this way intermediate-density lipoproteins (IDL), which are certainly derived in the main through VLDL catabolism. Further studies showed separate metabolic characteristics for the unbound and heparin-bound VLDL particles. Radiolabeled whole VLDL or unbound particles were reinjected into seven hypertriglyceridemic subjects and the kinetics studied in serial samples of plasma over the next 18-48 hours. The specific radioactivity-time curves of apoprotein B in the unbound and bound particles showed that the bound particles were derived wholly or partly from the unbound particles and in turn, were the precursors of IDL. This confirmed that heparin-bound VLDL particles represented VLDL undergoing catabolism, although in one subject about one-half of the bound particles appeared to have an origin other than through VLDL catabolism. These studies show that VLDL metabolism is heterogeneous, that the kinetics of total VLDL must be interpreted accordingly, and that the technique of heparin-affinity chromatography can be used for more detailed studies of VLDL.

Adult↗