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Biomedical subjects

M J Chapman

Publications and source records attributed to M J Chapman.

177 records · Page 10Linked to original sources
PubMed1976

Comparative immunochemical studies of the serum low-density lipoprotein in several animal species.

The antigenic reactivities of the serum LDLs of several groups of animals (mammals, birds, snakes, and fish) have been compared by qualitative and quantitative immunological techniques. The cross-reactivities of the various LDLs were evaluated qualitatively by gel diffusion techniques and quantitatively by the precipitation and inhibition of precipitation tests. The degrees of cross-reaction between human and animal LDLs ranged from 80-88% in the Old World monkeys to 36-58% in the pig, 26-37% in the guinea pig, 1-10% in the chicken, and about 1% in the snakes and fish. These antigenic relationships are consistent with the taxonomic classification of these species, but are relatively closer than those observed for other serum proteins (e.g., albumin, transferrin, and hemoglobin). These results indicate a relatively high conservation of LDL structure through evolution, and are consistent with a common function for this lipoprotein in the transport and metabolism of cholesterol in the higher animals.

Animals↗
PubMed1976

Low-density lipoproteins in patients homozygous for familial hyperbetalipoproteinaemia.

1. The low-density lipoproteins (LDL; density 1-007-1-063 g/ml) from two patients homozygous for familial hyperbetalipoproteinaemia have been submitted to chemical and physicochemical analysis. 2. The presence of an anomalous lipoprotein with a low proportion of triglyceride and a raised proportion of cholesterol has been confirmed. 3. In one patient, this lipoprotein accounted for about 85% of the LDL, but in the second, the amount varied from about 85% to a point at which it could not be detected among the coexisting normal lipoproteins. 4. The protein moiety of this anomalous LDL has effectively the same amino acid composition as that derived from the LDL of healthy subjects. 5. The proportions of carbohydrate, phospholipid and fatty acids could not be reliably distinguished from those of normal LDL. 6. The molecular weight and diffusion constant of the abnormal lipoprotein, even in the purest preparation, were close to the values determined for normal LDL of similar flotation rate.

Adult↗
PubMed1975

The distribution and partial characterization of the serum apolipoproteins in the guinea pig.

1. Very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins were isolated by sequential ultracentrifugation from the serum of male guinea pigs fed on a diet containing 3--4% fat. The apoproteins of these lipoproteins (apo-VLD, apo-LD and apo-HD lipoproteins) were studied after delipidation with organic solvents or extraction with tetramethylurea. 2. The major apolipoprotein of LD lipoprotein isolated by gel filtration was found to closely resemble apolipoprotein B of human serum in its chemical and physical properties. Electrophoresis in sodium dodecyl sulphate-polyacrylamide gel showed that this apoprotein consisted of a number of polypeptides. 3. Tetramethylurea precipitated an apoprotein from guinea-pig serum lipoproteins that is probably the apolipoprotein B-like component. This apoprotein accounted for about 80% of the apo-LD lipoprotein, about 55% of the apo-VLD lipoprotein and about 50% of the apo-HD lipoprotein. 4. The distribution of apolipoproteins soluble in tetramethylurea was determined by densitometric scanning of stained polyacrylamide disc gels. 5. A glycine-rich component of high electrophoretic mobility (band I) and a triplet of soluble apolipoproteins (bands II-IV) were present in both VLD and LD lipoprotein classes. These components constituted a higher proportion of the tetramethylurea-soluble apoproteins of VLD lipoprotein (60--80%) than of LD lipoprotein (40--55%). 6. Small amounts (10--15%) of a component of intermediate mobility, which contained traces of half-cystine, were also present in both VLD and LD lipoproteins. 7. A group of soluble components of basic character (bands VI-X), present as minor components of VLD lipoprotein (10--20%), constituted a major proportion (30--45%) of the soluble apoproteins of LD lipoprotein. Two of these apoproteins were rich in lysine, and two of lower electrophoretic mobility were rich in arginine. 8. The pattern of tetramethylurea-soluble apoproteins in HD lipoprotein was distinguished by the presence of two polypeptides of low electrophoretic mobility as its predominant components. One of these components, band VI, resembled the A-I apolipoprotein of man in both its amino acid profile and in its electrophoretic mobility. The second major component, band VI-B, was rich in lysine and resembled the C-I apolipoprotein of man in amino acid composition. 9. The soluble components of bands I and IX were analogous in physicochemical properties to the R-X1 and R-X2 (high-arginine polypeptide) peptides of human serum lipoproteins respectively.

Amino Acids↗
PubMed1973

The effect of a lipid-rich diet on the properties and composition of lipoprotein particles from the Golgi apparatus of guinea-pig liver.

1. A cell fraction rich in Golgi apparatus was isolated from the livers of guinea pigs fed on a lipid-rich diet (1.6% cholesterol, 15% corn oil). 2. The Golgi cisternae and secretory vesicles contained electron-dense particles which were tentatively identified as VLD (very-low-density) and LD (low-density) lipoproteins. Particles of moderate electron density, 150-500nm in diameter, were seen associated with membranous elements of the Golgi-apparatus cell fraction. Disruption of this cell fraction permitted the release of these three species of particles, which were separated into particulate lipid, and VLD and LD lipoproteins. 3. The large particles of moderate electron density, isolated as particulate lipid, were distinct from both species of Golgi particles in their chemical composition and in possessing an immunochemically unreactive apolipoprotein(s). Morphological observations suggest that the particulate lipid arose from cytoplasmic lipid droplets which were present as contaminants of the Golgi-rich fraction. 4. The chemical and immunochemical results are consistent with the suggestion that the Golgi LD particles are precursors of the VLD particles, into which they may be transformed by the addition of both triglyceride and cholesteryl ester. The present results provide further support for the proposal that the Golgi VLD particles are precursors of the serum VLD lipoproteins in the guinea pig. 5. Hepatic Golgi VLD particles isolated from guinea pigs fed on the lipid-rich diet contained significantly higher molar amounts (relative to protein) of both cholesteryl ester and triglyceride than similar particles from animals fed on a normal diet. These results suggest that the type of Golgi VLD particle produced from the LD particle is a direct consequence of the amount and composition of the dietary lipid. 6. Hepatic Golgi LD particles isolated from guinea pigs fed on different diets were similar in chemical composition and contained approx. 50% by weight of phospholipid. We conclude that the Golgi LD particle is normally present in the Golgi-apparatus cell fraction from guinea-pig liver, and may represent the end product of lipoprotein biosynthesis in the smooth endoplasmic reticulum. 7. The serum LD lipoproteins and Golgi LD particles were quite distinct in chemical composition. However, these two lipoprotein species were immunochemically identical and exhibited a similar range of flotation rate. It appears unlikely that the Golgi LD particles are secreted as the precursors of the serum LD lipoproteins.

Animals↗
PubMed1973

Localization of the enzymes of ketogenesis in rat liver mitochondria.

The localization of the enzymes of ketogenesis in isolated rat liver mitochondria has been investigated. Mitochondrial subfractions were isolated after disruption of this subcellular organelle by (a) hypotonic lysis in water, which permitted the ultracentrifugal separation of the soluble and membranous compartments of the mitochondrion, or by (b) a procedure involving swelling, contraction, and ultrasonic treatment, which permitted the isolation from discontinuous sucrose gradients of subfractions rich in intermembrane space protein, outer membrane, and inner membrane-matrix particles. Two membrane subfractions were invariably present as distinct bands at the lower interface of the discontinuous gradient. The upper of these two bands was found to be a highly purified preparation of outer mitochondrial membrane. Subfractions rich in matrix and in inner membrane were isolated from inner membrane-matrix particles after hypotonic treatment. The content of the various mitochondrial compartments in all subfractions was assessed from their enzymic and electron microscopic characteristics. The ketogenic activity of each subfraction was determined by measuring its capacity to form ketone bodies from acetyl CoA. The activity of this process was markedly enhanced by dithiothreitol. These measurements of ketone body formation, together with assays of individual enzymes of the ketogenic pathway, show that thiolase, HMGCoA synthase, and HMGCoA cleavage enzyme are localized in the matrix of the inner membrane-matrix particles. The rates of ketone body formation indicate that the HMGCoA synthase is the rate-limiting enzyme of the pathway in subfractions of high matrix content. Studies with sodium chloride indicate that a large portion of the HMGCoA synthase, which remains present in membrane subfractions derived from water-treated mitochondria, is bound by ionic interaction to component(s) of the membrane.

Acetyltransferases↗
PubMed1972

Lipoprotein particles from the Golgi apparatus of guinea-pig liver.

1. A cell fraction has been isolated from guinea-pig liver and shown to be rich in Golgi apparatus by electron microscopy. The activity of UDP-d-galactose-N-acetylglucosamine galactosyltransferase was over 100-fold greater in this cell fraction than in the liver homogenate. These data support the conclusion that the fraction was enriched in Golgi apparatus. 2. The Golgi cisternae and secretory vesicles contained electron-dense particles of 10-80nm diameter. Disruption of the Golgi apparatus cell fraction released these particles, which were separated into VLD (very-low-density) and LD (low-density) species on the basis of their density. 3. The Golgi VLD particles possessed morphological, flotational, chemical and immunochemical properties which closely resembled those of the serum VLD lipoproteins from the same animals. 4. The Golgi LD particles were rich in phospholipid, containing 48.1% by weight. The chemical composition of these particles was quite distinct from that of the serum LD lipoproteins, but did, however, show some similarity to that of the serum VLD lipoproteins. A marked resemblance was noted in the chemical characteristics of the Golgi LD and VLD particles (with the exception of triglyceride content). In addition, these two species of Golgi particles possessed the same antigenic determinant. 5. The results suggest that the Golgi VLD particles are the precursors of the serum VLD lipoproteins. On the basis of similarities in gross chemical composition and in the antigenic determinant of the Golgi LD and VLD particles, we conclude that the LD particles are probably the precursors of the VLD particles. In view of the marked differences in gross chemical composition of the Golgi LD particles and serum LD lipoproteins, it appears unlikely that the LD particles are directly secreted into the serum pool.

Animals↗
PubMedDate not supplied

Plasma lipoprotein and apolipoprotein profile in alcoholic patients with and without liver disease: on the relative roles of alcohol and liver injury.

In the present study, we report on alterations in plasma lipid, lipoprotein and apolipoprotein patterns in three separate populations of alcoholic patients, one without liver damage (Group I), a second presenting steatosis or mild alcoholic hepatitis or both (Group II) and a third with alcoholic cirrhosis (Group III), using a healthy, normolipidemic, nonalcoholic group as controls (Group C). Total plasma cholesterol levels were elevated in Groups II and III when compared with Groups I and C, while the ratio of esterified to free cholesterol was considerably lower in Group III than in the other groups. Plasma apo-AI levels were higher in Groups I and II than in Group C, but varied over a wide range in Group III. Apo-AII was present at higher concentrations in Groups I and II than in both Groups III and C. In contrast, no significant differences were detected in total apo-B levels, irrespective of the group. Modifications in the chemical composition of plasma lipoproteins primarily concerned a reduction in the cholesteryl ester content of low-density lipoproteins (LDL) and high-density lipoprotein (HDL) in Group III, this being compensated by a reciprocal increase in triglyceride. In addition, Group III lipoproteins, with the exception of HDL3 (density 1.100 to 1.140 gm per ml), exhibited a greater content of phospholipids than those of corresponding density from patients in Groups I and II. No significant differences were found in very low-density lipoprotein concentrations, while LDL levels increased in parallel with the severity of liver injury. In Groups I and II, HDL2 concentrations were elevated relative to Group C, while HDL3 decreased in parallel with the degree of impairment of liver function and thus from Group C to Group III.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMedDate not supplied

Comparison of the serum low density lipoprotein and of its apoprotein in the pig, rhesus monkey and baboon with that in man.

The principal form of the serum low density lipoprotein (LDL) in man, baboon, rhesus monkey and pig was isolated by preparative ultracentrifugation in the density interval 1.024-1.045 g/ml. The physicochemical characteristics of pig LDL most closely resembled those of man; thus, electrophoretic studies suggested that both baboon and rhesus LDL have a greater surface charge than that of their human counterpart, and electron-microscopic investigations showed baboon LDL (245 A) to be larger and rhesus LDL (205 A) smaller than those of man (217 A) and pig (228 A). In contrast, the immunological relationship between LDL from the two Old World monkeys and that of man was much closer (80-85% cross-reactivity by micro-immunoprecipitation) than that between pig and man (35% cross-reactivity). The principal difference between pig and human LDL appeared to reside in their protein and carbohydrate moieties. There was a marked resemblance between the protein moieties (apo-LDL) of LDL from the four species. The principal component of each animal apo-LDL was separated by gel-filtration chromatography and amounted to greater than 95% of the total protein; it exhibited a high molecular weight (greater than 250,000) upon SDS-polyacrylamide-gel electrophoresis and was indistinguishable from human apolipoprotein B in amino acid composition. Differences both between the apo-LDL and between the apo-B preparations from the four species, however, were detectable by immunological procedures. Such studies revealed inter-species relationships which were essentially the same as those observed between the respective native LDL preparations. The soluble apolipoproteins, present as minor components (less than 5%) of each apo-LDL, were compared by their electrophoretic mobility in polyacrylamide gel; the pattern seen in baboon and rhesus apo-LDL appeared to be most closely akin to that typical of their human counterpart. It is apparent that many characteristics typical of human serum LDL are found in those of the pig, rhesus monkey and baboon. Moreover, in view of the striking relationship existing between the immunological properties and apo-protein components of the LDL of the two Old World monkeys and that of man, these subhuman primates appear to be highly suitable as animal models for experimental atherosclerosis.

Amino Acids↗
PubMedDate not supplied

Expression of elastase activity by human monocyte-macrophages is modulated by cellular cholesterol content, inflammatory mediators, and phorbol myristate acetate.

We investigated the effects of a number of stimulatory agents on the production of both cell-associated and extracellular elastase-type enzymes on human monocyte-macrophages in vitro and of the modulation of such effects by modification of cellular cholesterol content. The stimulatory agents included phorbol myristate acetate (PMA) and the inflammatory mediators, lipopolysaccharide (LPS), opsonized zymosan (OZ), and platelet activating factor (PAF). Using the synthetic substrate, N-succinyl-trialanyl-paranitroanilide (SANA), we detected cell-associated elastase-like activity in monocyte-derived macrophages. Such activity increased markedly with cell maturation over the period from 5 to 15 days of adherence culture. While PAF (10 micrograms/ml) and LPS (10 micrograms/ml) were without effect on cell-associated elastase-like activity in macrophages, PMA (100 ng/ml) and OZ (1 mg/ml) markedly stimulated such activity in cells cultured for 15 days. Furthermore, a fivefold increase in the cell-associated elastase-like activity of macrophages occurred upon cholesterol loading of the cells with acetylated low density lipoprotein (AcLDL). By contrast, this activity was markedly diminished upon depletion of cellular cholesterol content after incubation with high density lipoprotein (HDL3). Latent elastinolytic activity in the culture medium was detected by use of a radioactive substrate, insoluble 3H-elastin, after initial tryptic treatment of the medium. Such latent elastase activity was secreted only by activated macrophages; the relative potency of stimulation was: PMA greater than LPS = PAF greater than OZ. Increase in cellular cholesterol content alone markedly enhanced the secretion of elastase (from undetectable levels to 28 ng of 3H-elastin degraded/hr/micrograms DNA). In all cases, both the cell-associated and secreted latent elastinolytic activities were due to metalloproteases, in view of their 90% inhibition by 2 mM EDTA. Cholesterol-loaded macrophages, which displayed an approximately 40-fold increase in total cholesterol content as compared to control cells, remained sensitive to the action of activators of OZ and PMA, while LPS and PAF exerted only weak effects. Our data indicate that cellular cholesterol content and inflammatory mediators are effective stimulants of the production and secretion of elastase-type enzymes by human monocyte-macrophages. Among these factors, cellular cholesterol content, OZ, PAF, and LPS may represent factors of relevance to the inflammatory role of the macrophage in atherogenesis and more specifically to the alteration of elastin structure in the extracellular matrix of the vessel wall.

Cells, Cultured↗
PubMedDate not supplied

Lipoprotein(a) and apolipoprotein(a) in a New World monkey, the common marmoset (Callithrix jacchus). Association of variable plasma lipoprotein(a) levels with a single apolipoprotein(a) isoform.

In an earlier report (Chapman et al, Biochemistry 1979;18:5096-5108), we suggested that the common marmoset may represent an important model for the study of human plasma lipoprotein metabolism. We now extend the interest of this monkey model to the study of lipoprotein(a) (Lp[a]) and apolipoprotein(a) (apo[a]). Density gradient ultracentrifugal fractionation of marmoset plasma revealed a bimodal distribution of Lp(a), with one peak of concentration occurring in association with very low density lipoproteins (VLDLs) and a second in the density range 1.040-1.080 g/ml. The dense Lp(a) subspecies displayed physicochemical properties (chemical composition, particle size, and electrophoretic mobility) that closely resembled those of its counterpart in humans and baboons but that were distinct from those of low density lipoprotein (LDL). Furthermore, the particle size of marmoset Lp(a) was invariant (31 nm) over the density interval 1.040-1.080 g/ml, whereas that of LDL decreased progressively with an increase in density (approximately 26-25.2 nm). Use of polyclonal and monoclonal antibodies to human apo(a) and of a polyclonal antibody to marmoset Lp(a) allowed immunologic identification of a single apo(a) isoform in the marmoset whose size was similar to that of apo B-100; apo(a) and apo B-100 were associated in Lp(a) particles by a disulfide linkage. The total protein mass of apo-Lp(a) was estimated to be 800,000 or more by electrophoresis in sodium dodecyl sulfate-polyacrylamide-agarose gels. The amino acid compositions of marmoset and human apo(a) resembled each other but were distinct from those of the corresponding forms of apo B-100. Immunologic evidence is provided for a high degree of cross reactivity between apo(a) in marmosets, baboons, and humans, supporting the idea of the existence of a marked degree of structural homology between these proteins. In addition, electroimmunoblotting of marmoset apo(a) and marmoset plasminogen showed that these proteins shared certain epitopes in common, suggesting that marmoset apo(a) may possess kringle-like structural features. Finally, despite possession of a single apo(a) isoform, marmoset Lp(a) levels varied over a 100-fold range (0.5-49 mg/dl plasma). Considered together, our present findings suggest that the common marmoset monkey constitutes a unique model in which to study the regulation of apo(a) gene expression and the posttranslational processing of apo(a), as well as factors that modulate the synthesis, intravascular metabolism, and cellular catabolism of Lp(a).

Animals↗
PubMedDate not supplied

[Matrix metalloproteinases and atherosclerosis. Therapeutic aspects].

Matrix metalloproteinases (MMPs) play a key role in the physiology of connective tissue development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. MMP family consists of at least 20 members; MMPs are produced by the different cell types (vascular smooth muscle cells, monocytes, endothelial cells) involved in the atheromatous plaque formation and participate to extracellular matrix remodelling and cell infiltration or migration. Since excessive tissue remodelling and increased matrix metalloproteinase activity have been demonstrated during atherosclerotic lesion progression (including plaque disruption), MMPs represent a potential target for therapeutic intervention to modify vascular pathology, by restoring the MMP/TIMP physiological equilibrium. This review highlights the structures of MMPs and their physiological inhibitors, the Tissue Inhibitors of MMPs (TIMPs), and describes the current developments in pharmacological MMP inhibition.

Animals↗