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Biomedical subjects

D S Ng

Publications and source records attributed to D S Ng.

At least 19 recordsLinked to original sources
PubMed1999

Targeted disruption of the murine lecithin:cholesterol acyltransferase gene is associated with reductions in plasma paraoxonase and platelet-activating factor acetylhydrolase activities but not in apolipoprotein J concentration.

Lecithin:cholesteryl acyltransferase (LCAT) deficiency resulting from targeted disruption of the Lcat gene in the mouse is associated with dramatic decreases in HDL concentration and the accumulation of nascent HDL in the plasma. We examined whether LCAT deficiency in mice is associated with a concomitant decrease in two antioxidative enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH). In control Lcat (+/+) mice both these enzymes are transported on HDL. Compared to Lcat (+/+) mice, HDL-cholesterol is reduced 94% and apoA-I, 90%, in Lcat (-/-) mice; this reduction in HDL is paralleled by a 71% decrease in PAF-AH activity and in a 58% decrease in PON activity. Apolipoprotein J (apoJ) levels, rather than being decreased, were significantly (P = 0.01) higher (36%) in Lcat (-/-) than in Lcat (+/+) mice, and the apo J/PON ratio was 3-fold greater in Lcat (-/-) than in Lcat (+/+) animals. Even though apolipoprotein A-I (apoA-I) concentration and PON activity were drastically reduced, there was no reduction in apoA-I and PON liver mRNA levels suggesting that post-transcriptional events are responsible for the reduction of plasma PON and apoA-I levels. Fast protein liquid chromatography (FPLC) revealed that in Lcat (+/+) mice both PON and PAF-AH activity is associated with large, apoA-I-containing HDL particles (9.7 nm by non-denaturing gradient gel electrophoresis) while in Lcat (-/-) mice both enzymes are associated with small 8.2 nm particles. We conclude that the concomitant reduction in HDL and apoA-I concentrations and PON and PAF-AH activities is best explained by rapid clearance of the small HDL particles found in LCAT deficiency.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

Disruption of the murine lecithin:cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I.

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Adrenal Glands↗
PubMed1996

Case report: retinopathy and neuropathy associated with complete apolipoprotein A-I deficiency.

Genetic deficiencies of plasma high-density lipoprotein cholesterol are associated variably with diseases of the eyes and nervous system. We ascertained a proband with undetectable plasma HDL-cholesterol due to homozygosity for a DNA mutation, APOA1 Q[-2]X, which encodes premature termination of translation of apolipoprotein in HDL. This person had a unique retinopathy, ataxia, and electrophysiologic abnormalities suggesting multifocal central nervous system deficits. Other gene carriers in this family had similar neurologic features, but only the proband had the retinopathy. The presence of retinopathy and neuropathy in affected family members was highly variable. This heterogeneity might results from time-dependent interactions with other genetic or environmental factors.

Adult↗
PubMed1995

Apolipoprotein A-I deficiency. Biochemical and metabolic characteristics.

Familial HDL deficiencies are associated with variable susceptibility to premature coronary heart disease, but the mechanism underlying this association remains poorly understood. Three homozygotes with isolated complete apo A-I deficiency caused by an autosomal codominant apo A-I Q[-2]X mutation and one heterozygote developed coronary heart disease before age 40 years. We characterized the effects of this mutation on lipoprotein metabolism. LDL FC, phospholipid, and apo B were all significantly higher in homozygotes than in heterozygotes. The HDLs of the heterozygotes were apo A-I poor relative to apo A-II. Lecithin-cholesterol acyltransferase activity was 59% lower in homozygotes than in normal subjects or heterozygotes. Cholesteryl ester transfer activity was increased in a homozygote compared with a normolipidemic control subject. Postprandial lipid metabolism was studied in one homozygote and one heterozygote. Post-prandial TG response in the homozygote was significantly exaggerated, while residual plasma HDL level remained unaffected. The homozygote also had delayed clearance of retinyl ester, a marker of chylomicron remnant metabolism. Thus, homozygosity and heterozygosity for apo A-I Q[-2]X are associated with qualitative, as well as quantitative, disturbances in plasma HDLs, LDLs, lipid-modifying enzyme activities, and postprandial retinyl ester metabolism. The observed elevation of atherogenic lipoproteins and reduction in antiatherogenic lipoproteins in the affected members of the apo A-I Q[-2]X kindred are consistent with the primary deficiency in apo A-I having pleiotropic effects that markedly enhance susceptibility for coronary heart disease.

Apolipoprotein A-I↗
PubMed1995

Expression of human lecithin-cholesterol acyltransferase in transgenic mice. Effect of human apolipoprotein AI and human apolipoprotein all on plasma lipoprotein cholesterol metabolism.

Human (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1.2- to 1.6-fold higher than that found in control mouse plasma. Lipid profiles, upon comparing Hu LCAT transgenics to control animals, revealed a 20 t0 60% increase in total and cholesteryl esters that were mainly present in HDL. The in vivo substrate specificity of Hu LCAT was assessed by creating animals expressing Hu apo AI + Hu LCAT (HuAI/ LCAT), Hu apo AI + Hu apo AII + Hu LCAT (HuAI/ AII/LCAT), and Hu apo AII + Hu LCAT (HuAII/LCAT). Plasma cholesterol was increased up to 4.2-fold in HuAI/ LCAT transgenic mice and twofold in the HuAI/AII/LCAT transgenic mice, compared with HuAI and HuAI/AII transgenic mice. HDL cholesteryl ester levels were increased more than twofold in both the HuAI/LCAT and HuAI/AII/LCAT mice compared with the HuAI, HuAI/AII, and HuLCAT animals. The HDL particles were predominantly larger in the HuAI/LCAT and the HuAI/AII/LCAT mice compared with those in HuAI, HuAII/LCAT, and HuLCAT animals. The increase in LCAT activity in the HuAI/LCAT and HuAI/AII/LCAT mice was associated with 62 and 27% reductions respectively, in the proportion of Hu apo AI in the pre beta-HDL fraction, when compared with HuAI and HuAI/AII transgenic mice. These data demonstrate that moderate increases in LCAT activity are associated with significant changes in lipoprotein cholesterol levels and that Hu LCAT has a significant preference for HDL containing Hu apo AI.

Animals↗
PubMed1994

Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia.

We report a Canadian kindred with a novel mutation in the apolipoprotein (apo) A-I gene causing analphalipoproteinemia. The 34-yr-old proband, product of a consanguineous marriage, had bilateral retinopathy, bilateral cataracts, spinocerebellar ataxia, and tendon xanthomata. High density lipoprotein cholesterol (HDL-C) was < 0.1 mM and apoA-I was undetectable. Genomic DNA sequencing of the proband's apoA-I gene identified a nonsense mutation at codon [-2], which we designate as Q[-2]X. This mutation causes a loss of endonuclease digestion sites for both BbvI and Fnu4HI. Genotyping identified four additional homozygotes, four heterozygotes, and two unaffected subjects among the first-degree relatives. Q[-2]X homozygosity causes a selective failure to produce any portion of mature apoA-I, resulting in very low plasma level of HDL. Heterozygosity results in approximately half-normal apoA-I and HDL. Gradient gel electrophoresis and differential electroimmunodiffusion assay revealed that the HDL particles of the homozygotes had peak Stokes diameter of 7.9 nm and contained apoA-II without apoA-I (Lp-AII). Heterozygotes had an additional fraction of HDL3-like particles. Two of the proband's affected sisters had documented premature coronary heart disease. This kindred, the third reported apoA-I gene mutation causing isolated complete apoA-I deficiency, appears to be at significantly increased risk for atherosclerosis.

Adolescent↗
PubMed1989

Effects of gold coordination complexes on neutrophil function are mediated via inhibition of protein kinase C.

Previous studies have shown that the gold compounds auranofin (AUR) and gold sodium thiomalate (GST) inhibit responses of various cells and tissues. We found that superoxide anion generation induced in human neutrophils by the chemotactic tripeptide fmet-leu-phe (1 microM), fluoride (18 mM), or phorbol myristate acetate (PMA, 100 nM) was inhibited by pretreatment of cells with 5-100 microM AUR. The extent of inhibition was dependent on AUR concentration and duration of the preincubation. GST was much less potent, inasmuch as only weak effects were observed at 5 times higher concentrations. The ineffectiveness of GST was attributed to its slower rate of penetration into cells, compared with AUR. The finding that mobilization of internal Ca2+ stores was not blocked in AUR-treated cells suggests that phospholipase C-mediated hydrolysis of polyphosphoinositides to inositol 1,4,5-trisphosphate was not inhibited by the drug. Because PMA is known to mimic the action of diacylglycerol in activating protein kinase C (PKC), we investigated the possibility that gold compounds might be interfering with signal transduction at this level. Enzymatic assays indicated that both gold compounds reduced the level of PKC activity associated with the cytosol; however, translocation of PKC to the plasma membrane was not found. Immunoblot analyses carried out with polyclonal anti-PKC antisera revealed that the gold compounds did not cause degradation of PKC or increase translocation to the membrane. Further studies indicated that enhanced endogenous protein phosphorylation resulting from PMA stimulation was attenuated in cells co-treated with AUR. Finally, in vitro enzymatic assays showed that both AUR and GST inhibited partially purified PKC in a concentration-dependent manner. It is suggested that modulation of PKC represents a mechanism of action of gold coordination complexes at the cellular level.

Auranofin↗
PubMed1989

Platelet-activating factor (PAF) stimulates phosphatidylinositol hydrolysis in human peripheral blood mononuclear leukocytes.

Human peripheral blood mononuclear leukocytes were labeled with myo-[3H]inositol and stimulated by platelet-activating factor (PAF). PAF-induced phosphatidylinositol hydrolysis and inositol-1,4,5-trisphosphate (IP3) formation in a dose-dependent manner. The response was very rapid and transient and parallels the time course of PAF-induced calcium mobilization in the same cells. However, the doses of PAF for inducing IP3 formation were much higher than those for calcium mobilization. PAF-induced IP3 formation was inhibited by PAF receptor antagonist, L-659, 989, but was not altered by either pertussis toxin or cholera toxin. These data suggest that PAF receptor in human peripheral blood mononuclear leukocytes may be coupled through a pertussis toxin-insensitive guanine nucleotide binding protein to a phosphoinositide-specific phospholipase C.

Calcium Channels↗
PubMed1989

Effect of platelet-activating factor (PAF) on cytosolic free calcium in human peripheral blood mononuclear leukocytes.

Cytosolic free calcium concentration, [Ca2+]i, in human peripheral blood mononuclear leukocytes was increased by platelet-activating factor (PAF) dose-dependently. The response was transient and was attenuated by EGTA (2 mM) in the medium. Furthermore, pretreatment of the cells with pertussis toxin did not alter significantly the rise in [Ca2+]i after stimulation by PAF. The data suggest that elevation of [Ca2+]i in human mononuclear leukocytes induced by PAF depends on: (1) calcium influx through calcium channels in the plasma membranes and (2) calcium mobilization from internal stores mediated via pathway(s) that is insensitive to the action of pertussis toxin.

Benzofurans↗
PubMed1988

Effect of sulfhydryl reagents on PAF binding to human neutrophils and platelets.

The effects of sulfhydryl reagents on the binding characteristics of platelet-activating factor (PAF) to PAF receptors in human neutrophils and platelets were examined. Simultaneous incubation of neutrophil or platelet membranes with radiolabeled PAF and N-ethylmaleimide (NEM), p-(chloromercuri)benzenesulfonic acid (PCMBS), or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in a dose-dependent inhibition of PAF specific binding to the membranes. PCMBS, a membrane-impermeable compound, also inhibited PAF specific binding to intact neutrophils and platelets. In the presence of PCMBS (0.1 mM) or NEM (20 mM), the ability of PAF to displace bound [3H]PAF from neutrophil membranes was reduced. Scatchard analysis of data from saturation binding experiments using neutrophil membranes indicated that the inhibitions of PCMBS (0.1 mM) and NEM (20 mM) on PAF specific binding were due to a decrease in the receptor affinity with no significant change in the number of binding sites. The results suggest that free sulfhydryl group(s) may be associated with the agonist binding site of PAF receptors in human neutrophils and platelets.

Blood Platelets↗
PubMed1988

Specific binding of platelet-activating factor (PAF) by human peripheral blood mononuclear leukocytes.

Binding of platelet-activating factor (PAF) to human peripheral blood mononuclear leukocytes was time-dependent, reversible, and saturable. [3H]PAF binding to the cells was inhibited dose-dependently by unlabeled PAF and PAF receptor antagonists: L-659,989, triazolam, and alprazolam. Scatchard analysis of saturation binding data indicated one class of receptors for PAF with KD = 5.7 nM and Bmax = 18 fmol/10(6) cells (11,100 receptors/cell). PAF (10 nM) increased intracellular free calcium concentration in human lymphocytes and this effect was inhibited by L-659,989 dose-dependently. Our data suggest that human peripheral blood mononuclear leukocytes have specific receptors for PAF.

Alprostadil↗
PubMed1988

Reactivity of acetoacetate with alkaline picrate: an interference of the Jaffé reaction.

Spectrophotometric, kinetic, and polarographic studies of the interaction of acetoacetate with alkaline picrate have been undertaken in the presence of aqueous NaOH concentrations ranging between 0.50 mol/L and 2.50 mol/L. Spectrophotometric data has substantiated formation of the following acetoacetate-picrate complexes: 1:1 red, 490 nm; 2:1 orange, 390 nm; and 3:1 colorless, 265 nm. Depending upon the time of measurement, the composition of alkaline picrate, and the acetoacetate level in the test samples, acetoacetate may be either a positive or negative interference in kinetic Jaffé methods for the determination of creatinine. Polarograms of alkaline picrate in 0.50 mol/L NaOH showed three well-defined nitro group reduction waves and a more diffuse fourth reduction wave with approximate half-wave potentials of -0.62 V, -0.79 V, -0.94 V, and -1.32 V, respectively. Increasing the concentration of hydroxide and/or acetoacetate resulted in the disappearance of reduction waves 1 to 3 with only reduction wave 4 remaining. Based upon the polarographic results, a trinitro anion structure has been assigned for the 2:1 acetoacetate-picrate complex.

Acetoacetates↗
PubMed1986

GTP regulation of platelet-activating factor binding to human neutrophil membranes.

Radiolabeled ligand binding studies showed that specific receptors for platelet-activating factor are present in human neutrophil membranes. GTP at 10(-7) to 10(-3) M decreased the specific binding of platelet-activating factor to neutrophil membranes in a dose-dependent manner. Inhibition of platelet-activating factor binding was also induced by other guanine nucleotides but not by adenine nucleotides. Our results suggest that platelet-activating factor receptor in human neutrophil membranes may be coupled to a guanine nucleotide binding protein.

Adenine Nucleotides↗
PubMed1986

High molecular weight forms of the insulin receptor.

The insulin receptor of liver, adipose, and placental plasma membranes was photoaffinity labeled with radioiodinated N epsilon B29-(monoazidobenzoyl)insulin. Three specifically labeled bands of 450, 360, and 260 kilodaltons (kDa) were identified in each tissue by polyacrylamide gel electrophoresis of the membranes solubilized in sodium dodecyl sulfate (SDS). The 360- and 260-kDa bands corresponded to partially reduced forms of the 450-kDa band. The distribution of radioactivity between the three insulin receptor bands was dependent on the tissue, the purity of the receptor preparation, and the conditions of solubilization in SDS. The 360- and 260-kDa bands became more prominent in each tissue with an increasing time of solubilization in SDS. However, with a short solubilization time in SDS, the 450-, 360-, and 260-kDa bands of the receptor were distributed approximately in a ratio of 85:15:0 in all three tissues. Inclusion of sulfhydryl alkylating reagents during solubilization in SDS altered this ratio to about 95:5:0. We conclude that the 450-kDa band represents the predominant form of the photolabeled insulin receptor and that the 260-kDa and probably the 360-kDa form as well were generated during the experimental manipulations preceding identification of the receptor. However, the appearance of the 360- and 260-kDa bands was not due to reductant present in SDS or buffer solutions and could not be accounted for by proteolytic degradation of the receptor. Furthermore, purification of the receptor over 2000-fold did not prevent the appearance of the 360- and 260-kDa bands.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1986

Jaffe' reaction products.

Polarographic and spectrophotometric studies of the interaction of creatinine with alkaline picrate have been undertaken in sodium hydroxide concentrations ranging between 0.95 and 4.5 mol/l. Red colored 1:1 and orange colored 2:1 creatinine-picrate complexes readily formed along with orange colored 1:1:1 creatinine-picrate-hydroxide complexes. The red and orange colored complexes were easily identified by their corresponding absorption maxima near 490 nanometers and 390 nanometers, respectively. Alkaline picrate polarograms showed three well-defined nitro group reduction waves with approximate half-wave potentials of -0.60 volts, -0.77 volts, and -0.91 volts. Increased concentrations of hydroxide and/or creatinine resulted in a decreased diffusion current for reduction waves 1-3 and the appearance of a fourth reduction wave, with an approximate half-wave potential of -1.24 volts. Further increases in base and/or creatinine concentration resulted in the disappearance of reduction waves 1-3, with only reduction wave 4 remaining. Based upon the experimental data, a tri-nitro anion structure has been assigned for the 2:1 complex.

Chemical Phenomena↗
PubMed1985

Peptide mapping of the insulin-binding site of the 130-kDa subunit of the insulin receptor by means of a novel cleavable radioactive photoprobe.

A radioactive photoaffinity probe for the insulin receptor was prepared by derivatizing insulin at its B29 lysine with a novel crosslinking reagent having a cleavable azo linkage. Insulin receptors purified from human placental membranes were photoaffinity labeled with this probe. The photolabeled receptor was treated with dithionite to cleave the azo linkage, thereby removing the insulin ligand and transferring the radioactivity to the receptor protein. The radioactive labeled subunit was isolated and digested with elastase for peptide mapping and separation by high performance liquid chromatography. Results obtained indicated that it will be feasible to use this new photoaffinity probe to obtain radioactive peptides representing the insulin-binding site(s) on the receptor subunit.

Affinity Labels↗
PubMed1985

Anti-morphine anti-idiotypic antibodies. Opiate receptor binding and isolated tissue responses.

Anti-idiotypic antibodies which recognize the opiate receptor were generated in guinea pigs following immunization against purified rabbit anti-morphine antibodies. The anti-idiotypic antibodies produced a concentration-dependent inhibition of [3H]naloxone binding to opiate receptors in a membranous mouse brain preparation. Saturation analysis indicated that the antibodies produced a non-competitive inhibition of naloxone binding. The ability of the antibodies to interact with biological systems was investigated in in vitro systems. In both the isolated guinea pig ileal longitudinal muscle and mouse vas deferens, the antibodies produced a concentration-dependent, opiate agonist-like action. The anti-morphine anti-idiotypic antibodies appear to interact specifically with the opiate receptor and may serve as useful tools in characterization of this receptor system.

Animals↗