Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusts migratoria) at rest and during flight

Summary Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoprotein epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.     

Long term incubation of cardiac myocytes with oleic acid and very-low density lipoprotein reduces heparin-releasable lipoprotein lipase activity

An exogenous [³H]triolein emulsion was hydrolyzed by intact cardiac myocytes with functional LPL located on the cell surface. This surface-bound LPL could be released into the medium when cardiac myocytes were incubated with heparin. Incubation of cardiac myocytes with VLDL, or the products of TG breakdown, oleic acid or 2-monoolein, did not increase LPL activity in the medium. However, incubation of cardiac myocytes with either VLDL or oleic acid for > 60 min did reduce heparin-releasable LPL activity. In the heart, this inhibitory effect of FFA could regulate the translocation of LPL from its site of synthesis in the cardiac myocyte to its functional site at the capillary endothelium.Abbreviations LPL lipoprotein lipase - TG triacylglycerol - FFA free fatty acids - VLDL very-low density lipoprotein     

Lipolysis of serum-activated triacylglycerol at the surface of J774.1 macrophages

Summary Cultured mouse (J774.1) macrophages accumulated triacylglycerol, but no cholesteryl ester or cholesterol, when incubated in albumin-poor medium with serum-activated lipid particles containing 84 mol% trioleoylglycerol and 9 mol% cholesteryl oleate. Accumulation of triacylglycerol by cells was associated with hydrolysis of particulate triacylglycerol to fatty acid and glycerol. Both acyl and glyceryl moieties of particulate triacylglycerol were recovered in cellular triacylglycerol with a molar ratio of 3.6. The cells also accumulated fatty acid and monoacylglycerol. Whether acylglycerol was taken up as a single molecular species, such as monoacylglycerol, or as several species can not be determined by the present findings. Macrophages incubated with lipid particles for 24 h had many lipid particles attached to cell surfaces and numerous intracellular lipid droplets. The surface film of attached particles was continuous with the outer leaflet of plasma membrane of the cells. Particles partially depleted of core triacylglycerol and collapsed surface films were found attached to surfaces of macrophages. There was no morphological evidence that lipid particles were taken up intact by cells, through endocytosis or phagocytosis. Macrophages incubated with lipid particles also contained intracellular lamellar structures. They varied in size and shape, and were located in the periphery of cells, sometimes near lipid droplets and endoplasmic reticulum. Only 3% of the lamellar structures were associated with lysosomes, indicating they probably were not of lysosomal origin. Lipid particles attached to cells decreased in size and number, and lamellar structures developed at the surface of particles, or replaced the particles, when glutaraldehyde-fixed specimens were incubated at 25° C, demonstrating lipolytic activity at the surface of macrophages. Our findings suggest that particulate triacylglycerol was hydrolyzed by lipoprotein lipase at the surface of macrophages, and that fatty acid and monoacylglycerol formed by lipolysis were transported directly into the cells to be reesterified. When lipolytic products were taken up faster than they could be utilized, they accumulated as lamellar structures in the cells.Abbreviations MEM Eagle's alpha modification of minimum essential medium     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

北京鸭血清HDL主要载脂蛋白的高效液相层析分离及其理化性质

用高效液相层析(HPLC)对北京鸭血清高密度脂蛋白(HDL)的所有载脂蛋白(apo)分离纯化,得五个主要apo峰,经SDS-PAGE鉴定,均为单一带;并做了IEF、糖基含量分析;对其中含量高的四个apo峰样进行了氨基酸组成测定和N-端部分氨基酸序列分析;从所做理化性质的研究,发现北京鸭血清HDL中的主要apo为:A-Ⅰ、C-Ⅲ_o、apoC-Ⅲ_s(s=1,2)、apoC's及可能的A-Ⅲ,几乎不含E和A-Ⅱ_o北京鸭HDL中apo的上述组成特点明显不同于易患动脉粥样硬化(As)的人及兔等动物,在一定程度上决定了北京鸭HDL的形成与代谢具有其特点,该特点与北京鸭不易形成As密切相关。     

巨噬细胞对正常人极低密度脂蛋白亚组分代谢的研究

本文研究了小鼠腹腔巨噬细胞对正常人极低密度脂蛋白(N-VLDL)两种亚组分VLDL_1和VLDL_3的代谢。两种亚组分都能以受体方式和非特异性方式被巨噬细胞摄取和降解。在受体途径中以VLDL_1的摄入量居多。对胞内甘油三酯(TG)的堆积作用以VLDL_1较强,对胆固醇酶(CE)的堆积则以VLDL_3较强。表明两者在促进巨噬细胞向泡沫细胞转变中的作用有所不同。     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight     

The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13

Abstract The pCloDF1S encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murein lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF13.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate