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H Robenek

Publications and source records attributed to H Robenek.

At least 37 records · Page 2Linked to original sources
PubMed1997

Interaction of lipoproteins with type II pneumocytes in vitro: morphological studies, uptake kinetics and secretion rate of cholesterol.

Apart from dipalmitoyl phosphatidylcholine, cholesterol is the most abundant surfactant lipid. About 90 to 99% of cholesterol of the alveolar surfactant is derived from serum lipoproteins. The aim of this study was to identify the lipoprotein which preferentially supplements type II pneumocytes with cholesterol destined for surfactant production. Ultrastructural investigations revealed that type II pneumocytes bind and take up HDL, LDL and VLDL. Binding and uptake of VLDL occurred even in the presence of excess LDL indicating that, besides LDL receptors, type II pneumocytes express additional binding sites for VLDL. Type II pneumocytes in primary culture are able to take up cholesterol added in the form of HDL, LDL and VLDL. Cholesterol uptake was lowest from HDL and highest from VLDL. The maximal velocity of cholesterol uptake from VLDL was more than three times that of cholesterol uptake from LDL. The half-maximal saturation of cholesterol uptake from VLDL was nearly half that of LDL. From these kinetic data and the distribution of free cholesterol among the serum lipoproteins, we calculated that the cholesterol uptake from VLDL is more than three times that of cholesterol uptake from LDL. In double-labeling experiments type II pneumocytes secreted palmitic acid-labeled phospholipids together with labeled free cholesterol taken up from lipoproteins. The secretion rates of both phospholipids and free cholesterol were stimulated to nearly the same extent by isoproterenol. From our results we conclude that type II pneumocytes interact specifically with HDL, LDL and VLDL. Cholesterol taken up in the form of the individual lipoproteins shows no difference in its availability for the formation of cholesterol ester and surfactant by type II pneumocytes in vitro. Based on the kinetic studies, it appears that VLDL is the major gateway through which cholesterol is provided to satisfy the cholesterol requirements of type II pneumocytes for the synthesis of surfactant.

Animals↗
PubMed1996

Pyrogallol red-vanadium complex-a new stain for electron microscopy.

We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (lambda max = 520 nm) to violet (lambda max = 539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultratain sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to protein-aceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level.

Animals↗
PubMed1996

Differences in the mechanisms of uptake and endocytosis of small and large chylomicron remnants by rat liver.

Initial binding and subsequent endocytosis of small and large chylomicron remnants by rat liver were compared. Small and large chylomicrons were obtained from mesenteric lymph of glucose- or fat-fed rats, respectively. The low-density lipoprotein (LDL) receptor was up- and down-regulated as shown by LDL receptor messenger RNA (mRNA). The rate of removal of small chylomicron remnants by isolated perfused rat livers followed closely the activity of the LDL receptor. When mRNA was undetectable, the uptake was as low as that of lymphatic small chylomicrons. In contrast, the uptake of large chylomicron remnants into perfused rat livers was unaffected by changes of the LDL-receptor activity, but significantly reduced after livers were flushed with heparin or heparinase. Large chylomicron remnants were cleared from plasma much faster than small chylomicron remnants, but were more slowly internalized into hepatocytes. Both, small and large chylomicron remnants entered the pathway of receptor-mediated endocytosis as shown by electron microscopy and analysis of isolated endosomes. Yet, large chylomicron remnants were taken up into the compartment of uncoupling of receptors and ligands and multivesicular bodies at a much slower rate. This was independent of the activity of the LDL receptor and the heparin-releasable binding site. From these findings it is concluded that large chylomicron remnants initially bind rapidly to surface components other than the LDL receptor, one of which may be hepatic lipase. Yet, the consecutive internalization is slow. In contrast, small chylomicron remnants are removed at a slower rate from plasma, binding predominantly to the LDL receptor, but are more readily taken up into endosomes.

Animals↗
PubMed1996

Effects of calcium on the migration and recruitment of macrophages and macrophage-derived foam cells.

Calcium is thought to play an important role in the genesis of atherosclerotic lesions, but the precise mechanisms involved are unclear. In the present investigation, we have used in vitro systems to investigate the effects of calcium on one key aspect of lesion development: the migration of macrophages and macrophage/foam cells. Using agarose plate migration assays, the migratory characteristics of macrophages exposed to 1) no lipoprotein, 2) low density lipoprotein (LDL), 3) acetylated low density lipoprotein (acLDL), and ) oxidized low density lipoprotein were examined. The most marked stimulatory effect on macrophage mobility was observed when freshly isolated cells were exposed to acLDL during the migration assay. High levels of exogenous calcium were found to suppress the stimulatory effect of acLDL on migration. As the responses of macrophages exposed to a uniform concentration of agents in the surrounding medium may differ from chemotactic responses to a concentration gradient, the migration of macrophages, with and without preexposure to acLDL or LDL, was studied using microchemotaxis Boyden chambers. Under these conditions, calcium acted as a highly potent chemoattractant, especially to cells that had been preincubated with acLDL. These results suggest how elevated external calcium concentration leads initially to macrophage recruitment, and subsequently to foam cell aggregation and lipid core formation, in association with calcification, in the developing atherosclerotic plaque.

Animals↗
PubMed1996

Endocytic mechanisms for uptake and metabolism of chylomicron remnants in the liver.

Removal of small chylomicron remnants by perfused rat livers closely correlates with the LDL-receptor mRNA modulated by various interventions. In contrast, removal of remnants of large chylomicrons is not appreciably influenced by the activity of the hepatic LDL-receptor. Their primary removal depends on a heparinase-sensitive binding site. Transient and stable transfection of the cDNAs of the two subunits of the human asialoglycoprotein receptor markedly increased the binding capacity for chylomicron remnants suggesting the asialoglycoprotein receptor to be an alternative mechanism for remnant removal. Some species can edit apolipoprotein B-100 mRNA and thus secret apolipoprotein B-48 containing lipoproteins of hepatic origin. Generally these animals have lower cholesterol levels and a more favorable lipoprotein profile.

Animals↗
PubMed1996

Interaction of apo E-containing lipoproteins with the LDL-receptor-related protein, LRP.

The low density lipoprotein receptor-related protein (LRP) discovered in 1988 (1) was proposed as a candidate for the postulated apo E-binding chylomicron remnant receptor. Recent results suggest LRP to be a multifunctional cell surface receptor which might have a pivotal function in linkage of diverse metabolic pathways not previously considered to be related. Although biochemical evidence for lipoprotein binding to LRP has been presented (rev in 2), the need to add apo E to lipoproteins to demonstrate lipoprotein binding to LRP has raised doubts as to its lipoprotein receptor function. Therefore, it has yet to be established whether there is a physiologically relevant binding of naturally occurring apo E-containing lipoproteins to LRP. Here we describe cytochemical studies in which naturally occurring apo E-containing lipoproteins carefully isolated by gelfiltration chromatography were found to bind specifically to the cell surface of mouse peritoneal macrophages (MPM). These lipoproteins compete specifically with the binding of activated alpha 2-macroglobulin, which is a generally accepted ligand of LRP, to these cells without previous enrichment with exogenous apo E. We therefore conclude that LRP is indeed a physiologically relevant lipoprotein receptor.

Animals↗
PubMed1996

Influence of native and modified lipoproteins on migration of mouse peritoneal macrophages and the effect of the antioxidants vitamin E and Probucol.

Macrophage-derived foam cells are a key component of the atherosclerotic plaque. Stimulation of the migration of foam cells back to the blood potentially offers an approach to plaque regression. However, information on the natural and synthetic factors influencing macrophage migration remains limited. The aim of the present study was to determine the effects of different lipoproteins and antioxidants on the migration of mouse peritoneal macrophages and macrophage-derived foam cells. The migration of cells was determined in vitro by using a Boyden chamber. Cellular content of cholesterol and cholesteryl esters was measured by HPLC. The native lipoproteins, VLDL, LDL, and HDL, and the chemically modified lipoproteins acetylated LDL (acLDL) and oxidized LDL (oxLDL), all acted as chemoattractants for macrophages that had not been lipid-loaded. The antioxidants vitamin E and Probucol had chemoattractive and chemokinetic effects on the migration of unloaded mouse peritoneal macrophages. Foam cells (lipid-laden macrophages) showed decreased mobility with increased cellular lipid content. However, as macrophages accumulated higher amounts of cholesterol and cholesteryl esters, the additional decrease of cell migration was relatively minor. Simultaneous incubation of macrophages with acLDL and vitamin E or Probucol increased migration. The content of cellular cholesterol and cholesteryl esters was not changed when acLDL-treated cells were exposed at the same time to vitamin E, but cholesteryl ester content was increased in the presence of Probucol. Overall, the findings indicate that the antioxidants vitamin E and Probucol have a potential anti-atherogenic action in stimulating migration of lipid-laden macrophages without major impairment of the ability of the cells to accumulate and metabolize modified LDL.

Animals↗
PubMed1996

Measurement of small high density lipoprotein subclass by an improved immunoblotting technique.

An improved method of immunoblotting of plasma onto agarose gel matrix containing antiapolipoprotein A-I is described. Fresh plasma samples were subjected to gradient polyacrylamide gel electrophoresis (4-25%) followed by electrotransfer onto agarose gel layer containing antiapolipoprotein A-I. This method was compared with immunoblotting onto nitrocellulose where the transfer onto agarose gel matrix has been shown to be more convenient, quantitative, and can be kept permanently. Plasma apolipoprotein A-I was found to be distributed among regions of varying molecular weights ranging from 43,000 to 800,000. A small size fraction of molecular weight range of 43,000-50,000 (small HDL) was found in normolipidemic and hyperlipidemic subjects. The proportion of the latter fraction varied considerably among subjects (range: 0.0-32%), being lower in normolipidemic subjects (mean +/- SEM: 11.6 +/- 1.4%), and higher in hyperlipidemic subjects (mean +/- SEM: 23.7 %/- 1.7%, P < 0.001). Physiological increase in the level of the small HDL was observed in normolipidemic subjects 4 h after fat ingestion (difference: 5.0%, P < 0.001); moreover, the level was higher in normolipidemic subjects who consumed moderate amounts of alcohol (mean +/- SEM: 17.9 +/- 1.2%, P < 0.001) compared with normolipidemic subjects who do not drink alcohol at all.

Antibodies↗
PubMed1995

Interaction of biglycan with type I collagen.

The small proteoglycan decorin is known to interact with type I collagen fibrils, thereby influencing the kinetics of fibril formation and the distance between adjacent collagen fibrils. The structurally related proteoglycan biglycan has been proposed not to bind to fibrillar collagens. However, when osteosarcoma cells were cultured on reconstituted type I collagen fibrils, both decorin and biglycan were retained by the matrix. Immunogold labeling at the electron microscopic level showed that both proteoglycans were distributed along collagen fibrils not only in osteosarcoma cell-populated collagen lattices but also in human skin. Reconstituted type I collagen fibrils were able to bind in vitro native and N-glycan-free biglycan as well as recombinant biglycan core protein. From Scatchard plots dissociation, constants were obtained that were higher for glycanated biglycan (8.7 x 10(-8) mol/liter) than for glycanated decorin (7 x 10(-10) mol/liter and 3 x 10(-9) mol/liter, respectively). A similar number of binding sites for either proteoglycan was calculated. Recombinant biglycan and decorin were characterized by lower dissociation constants compared with the glycanated forms. Glycanated as well as recombinant decorin competed with glycanated biglycan for collagen binding, suggesting that identical or adjacent binding sites on the fibril are used by both proteoglycans. These data suggest that, because of its trivalency, biglycan could have a special organizing function on the assembly of the extracellular matrix.

Biglycan↗
PubMed1995

Immunocytochemical localisation of some lysosomal hydrolases, their presence in luminal fluid and their directional secretion by human epididymal cells in culture.

The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid alpha-glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, beta-hexosaminidase (N-acetylglucosaminidase) and alpha-glucosidase were measurable in the luminal fluid from the human corpus epididymidis; beta-hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with 35S-methionine revealed that the precursors of cathepsin D and beta-hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.

Adult↗
PubMed1995

Repression of the macrophage scavenger receptor in macrophage-smooth muscle cell heterokaryons.

Macrophage scavenger receptors mediate the uptake of chemically modified LDL in an unregulated manner, leading to massive intracellular accumulation of lipid and thus a foamy cellular morphology. In atherosclerotic lesions, foam cells originate not only from macrophages but also from smooth muscle cells, yet smooth muscle cells do not normally express scavenger receptors, and when exposed to chemically modified LDL in vitro, lipid accumulation does not occur. The mechanism of conversion of smooth muscle cells into foam cells in the arterial wall is thus still under discussion. To investigate whether direct interaction between macrophages and smooth muscle cells may be involved and to explore the effects of components of the two cell types on the expression of scavenger receptors, we report here experiments using somatic cell hybrids formed by fusion of the two cell types. Immunofluorescent labeling and confocal microscopic techniques were applied to investigate and measure (1) lipid accumulation (using Nile Red staining), (2) the binding and uptake of acetylated LDL (using 1,1'-dioctadecyl-1-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated LDL), and (3) receptor expression (assessed using a specific anti-receptor antibody) in smooth muscle cell-macrophage heterokaryons, macrophage-macrophage homokaryons, smooth muscle cell-smooth muscle cell homokaryons, and unfused macrophages and smooth muscle cells. The results demonstrate that scavenger receptor expression becomes repressed in macrophage-smooth muscle cell heterokaryons but not in macrophage-macrophage homokaryons. One possible explanation for the observed repression would be the existence of a negative regulatory cytoplasmic factor produced by smooth muscle cells.

Animals↗
PubMed1995

Kringle 4 of human apolipoprotein[a] shares a linear antigenic site with human catalase.

Monoclonal antibody (mab) 1A2, directed against human apolipoprotein[a] (apo[a]), revealed a strong reaction with peroxisomes as shown by immuno-gold labeled cryosections of human liver biopsies. This reactivity was not due to the presence of apo[a] in peroxisomes but to a cross-reactivity of mab 1A2. Immunoblot analysis of peroxisomal fractions and purified human catalase demonstrated that mab 1A2 reacts with catalase. Conversely, an anti-catalase antibody also recognized apo[a]. By sequence comparison we identified a 4-amino acid motif (Y-Y-P-N) that is shared between the highly repetitive kringle 4 motif of apo[a] and the carboxy-terminal third of the peroxisomal marker enzyme catalase. No other identical sequences were identified in these proteins. Results from the following experiments indicated that 1A2 recognizes this short linear epitope. i) Mab 1A2 reacted only with the 4 amino acid peptide sequence in a pin-ELISA using immobilized overlapping peptides. ii) A synthetic peptide including this sequence completely inhibited the 1A2 immunoreactivity to apo[a] and catalase. iii) A recombinant fusion protein tagged with the putative epitope was recognized by mab 1A2. Our findings demonstrate that unknown linear epitopes in native proteins can be identified by sequence comparison between known proteins. The practical implication is that antibodies against apo[a] must be controlled for this cross-reactivity before using them for immunohistochemical studies of intracellular apo[a] in tissues or cells.

Amino Acid Sequence↗
PubMed1995

Inhibition of smooth muscle cell proliferation and neointimal growth by low-anticoagulant heparin.

Low-anticoagulant heparin (LA-heparin) obtained by affinity chromatography on antithrombin III Sepharose inhibits the proliferation of cultured arterial smooth muscle cells in an in vitro bioassay system as effectively as standard heparin. A growth inhibition of smooth muscle cells of about 60% is achieved when LA-heparin or heparin is added to the culture medium to a concentration of 50 micrograms/ml. In normolipemic rats LA-heparin suppresses the formation of neointimal thickenings and stenosis after balloon catheter-induced deendothelialization of the carotid artery. In terms of mass a dose of 5 mg/kg body weight/d given subcutaneously twice daily one week before and 2 weeks after balloon injury the cross sectional area of the neointima is reduced to 36% as compared with the nontreated control group (100%). This 64% reduction is statistically highly significant (p < 0.001). After treatment with 0.5 mg LA-heparin/kg/d the reduction of the neointima was 11% (p < 0.05). At a dose of 5 mg/kg body weight single or repeated administrations of LA-heparin caused only a small and transient increase in activated partial thromboplastin time values. The results show that subcutaneous administration of LA-heparin very effectively prevents smooth muscle cell proliferation and balloon catheter-induced neointimal growth. The well tolerated systemic application of this chemically non-modified LA-heparin might justify clinical trials for prevention of restenosis after percutaneous transluminal coronary angioplasty or other invasive cardiovascular interventions without complications of bleeding.

Angioplasty, Balloon↗
PubMed1994

Demonstration of Sindbis virus antigen in Lowicryl-embedded cultured cells by immunogold staining.

Eight monoclonal antibodies (MAbs) were tested for the use in immunogold staining of Sindbis virus (SIN) antigen in cultured Baby Hamster kidney (BHK) cells. The antibodies were directed against the capsid (C) protein (5) or against the glycoprotein E1 (3), respectively. Four out of five anti-C MAbs and two out of three anti-E1 Mabs reacted on frozen sections, whereas two of the anti-C antibodies and none of the anti-E1 antibodies reacted on Lowicryl K4M-embedded cells. The MAbs reacting, in particular the anti-C Mabs which react on Lowicryl K4M-sections, will allow further studies of the pathogenesis and replication of alphaviruses on the cellular level. The results also confirm the conformation-dependence of the glycoprotein epitopes.

Acrylic Resins↗
PubMed1994

Receptor-mediated endocytosis is sensitive to antibodies against the uncoating ATPase (hsc70).

We have investigated the functional role of the coated vesicle-uncoating ATPase (UA), a cognate heat shock protein (hsc70), in receptor-mediated endocytosis. A monoclonal antibody against bovine brain UA/hsc70 was generated that recognizes a 26 kDa proteolytic fragment harbouring the putative clathrin-binding site. In vitro, this antibody blocked the UA/hsc70-mediated release of clathrin from isolated coated vesicles (CVs). Upon microinjection into tissue culture cells, it specifically inhibited the heat shock-induced nuclear migration of UA/hsc70. This antibody also interfered with endocytosis of ligand-receptor complexes in injected cells. Two different systems were studied: the uptake of aggregated human IgG by BHK cells transfected with a human Fc receptor (FcRII), and the internalization of LDL by human fibroblasts. Injection of the monoclonal antibody in concentrations yielding approximately equal molar ratios of antibody to enzyme resulted in a reduction of endocytosis to 20-30% of control values, as seen by conventional light and confocal laser scanning microscopy, and by electron microscopy. In the transfected BHK cells, the endocytosed ligand remained associated with the labeling for clathrin and was not delivered to the endosomal compartment within the period expected from control serum- or non-injected cells. Thin sections revealed an accumulation of coated structures in the antibody-injected cells as compared to controls. Thus, our data show that UA is essential for normal receptor-mediated endocytosis, and is presumably involved in the uncoating of CVs preceding their fusion with endosomes.

Animals↗