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H Robenek

Publications and source records attributed to H Robenek.

At least 91 records · Page 5Linked to original sources
PubMed1986

Polypeptides of the chloroplast envelope membranes as visualized by immunochemical techniques.

The polypeptides of relative molecular masses (Mr) 22,000, 29,000, and 36,000 represent three major constituents of the chloroplast envelope of spinach (Spinacia oleracea L.) leaves. The Mr 22,000 polypeptide has been localized in the outer membrane, whereas the two other peptides have been attributed to the inner envelope membrane (Joyard et al., 1983). The Mr 29,000 polypeptide has been identified as the "phosphate translocator" (Flügge and Heldt, 1979). In this investigation, we studied the three envelope polypeptides by means of immunocytochemistry. Using indirect immunofluorescence, all three polypeptides were visualized in cryostat sections of formaldehyde-fixed leaf tissue. They were found in both palisade and spongy parenchyma cells and in guard cells, as indicated by a strong fluorescence in the chloroplast periphery. In contrast, fluorescein isothiocyanate or protein A-gold labeling of isolated fixed chloroplasts resulted only in visualization of the Mr 22,000 polypeptide, a constituent of the outer membrane. We further studied the morphological distribution and frequency of this peptide by electron microscopic evaluation of platinum-carbon replicas after freeze-etching or label-fracture and of ultra-thin sections. By use of these three methods, the polypeptide was found to be randomly distributed in the outer envelope membrane and easily accessible to the immunomarker. Average marker density, as obtained by freeze-etching and label-fracture, was approximately 130 gold particles per square micron.

Chloroplasts↗
PubMed1986

Apolipoprotein B determination in the dissolved precipitate obtained after precipitation of LDL with polyvinylsulphate. An alternative method for the determination of LDL apolipoprotein B without using ultracentrifugation.

Using a commercially available test for LDL cholesterol (Boehringer Mannheim), a method was developed for determination of LDL apolipoprotein B without using ultracentrifugation. The infranatant obtained by precipitation of serum with polyvinylsulphate was redissolved in a saline citrate solution and incubated with phospholipase A2, phospholipase C, phospholipase D or triglyceride lipase, respectively. When the saline-citrate redissolved precipitate was monitored by electron microscopy, it appeared as a fibriform network. The additional incubation with phospholipase A2, C, D, or triglyceride lipase resulted in a molecularization of LDL particles. By electron microscopy these particles could not be distinguished from LDL particles isolated by ultracentrifugation. In radial immunodiffusion tests, the additional incubation of the redissolved precipitate with phospholipase C or phospholipase D resulted in a total loss of the slight immunoreactivity observed before phospholipase incubation. However, additional incubation with triglyceride lipase resulted in a significant increase in immunoreactivity. Only the additional incubation of the redissolved precipitate with phospholipase A2 resulted in an immunoprecipitation reaction comparable to that with LDL particles isolated by ultracentrifugation. Using a resolubilized and phospholipase A2-incubated precipitate of a pool serum as apolipoprotein B standard, a good correlation was obtained between apolipoprotein B values measured in this dissolved precipitate and those measured in the d greater than 1.006 kg/l fraction isolated by ultracentrifugation (r = 0.95; y = 0.95x + 0.018; n = 44).

Apolipoproteins B↗
PubMed1986

Detection of localization and internalization of membrane DNA in the Tetrahymena by the lactoferrin--colloidal gold technique.

It is known that lactoferrin binds to DNA. In the present study a lactoferrin-gold complex bound initially to the ciliary membrane, then appeared in coated pits and intracytoplasmic vesicles. On treatment for 1 h, the gold particles could be detected near to the nucleus. The lactoferrin-gold complex neither bound to the surface, nor appeared in the cytoplasm of those cells which had been exposed to DNAse for pretreatment.

Animals↗
PubMed1985

Interaction of high density lipoproteins with cholesteryl ester-laden macrophages: biochemical and morphological characterization of cell surface receptor binding, endocytosis and resecretion of high density lipoproteins by macrophages.

Morphological and biochemical experiments were carried out to investigate the interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages. It is demonstrated that resident mouse peritoneal macrophages express HDL receptors. Subsequent to receptor-mediated binding, HDL are internalized and intracellularly transported into endosomes. These endosomes do not fuse with the lysosomal compartment but interact with the margin of intracellular plasma lipid droplets. Macrophages do not degrade, but rather resecrete internalized HDL particles as described for the transferrin-receptor pathway. HDL binding to freshly isolated macrophages is saturable at a concentration of approximately 320 ng HDL-protein/mg cell protein and a Scatchard plot indicates the presence of some 130 000-190 000 receptors/cell with a Kd of approximately 9 X 10(-7) M. Binding of HDL on the macrophage surface is significantly enhanced in cholesterol-laden macrophages, whereas the increase in the rate of uptake and secretion is less pronounced. Within the HDL fraction the HDL2 subclass showed higher binding, uptake and secretion activity as compared with HDL3. From these experimental data we postulate that cholesterol uptake from macrophages is mediated by HDL particles which interact with these cells via a receptor-mediated retroendocytosis pathway.

Animals↗
PubMed1985

Primary culture of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. V. Disturbance of the cellular membrane and bile canalicular ultrastructure induced by chlorpromazine.

In the present paper rat hepatocytes in primary monolayer culture were used to investigate the adverse effects of chlorpromazine (CPZ) at the cellular level. As revealed by thin sectioning many of the ultrastructural alterations were comparable to those described for the isolated perfused rat liver under the influence of CPZ. Alterations comprised short-term effects, such as dilation of the rough endoplasmic reticulum and the nuclear envelope, and long-term effects including huge accumulations of myeloid bodies within the cytoplasm as well as dilation and diverticulation of bile canaliculi. Freeze-fracturing revealed the dislocation of intramembrane particles in the sinusoidal plasma membrane which could be detected as early as 30 min after exposure to CPZ. As judged from filipin cytochemistry, alterations in the cholesterol content seems to play a minor role in the process of membrane damage except at the sinusoidal surface where a reduction of cholesterol content may contribute to the impairment of membrane functions. It is concluded that CPZ exerts its cholestatic effect primarily by a rapid disturbance of the membrane architecture of the sinusoidal surface and secondarily by other interactions with the bile secretory apparatus.

Animals↗
PubMed1985

Proliferation, morphology, and low-density lipoprotein metabolism of arterial endothelial cells cultured from normal and diabetic minipigs.

Aortic endothelial cells from control and streptozotocin diabetic minipigs were cultured. Both groups of cells exhibited the typical cobblestone-like appearance and gap junction formation. Endothelial cells derived from diabetic minipigs differed, however, from those from control animals by a higher rate of proliferation and a higher percentage of large and often multinucleated cells. In these cells the specific binding of low-density lipoproteins (LDL) to coated pits on the cell surface, the LDL uptake, and the intracellular transport of LDL to lysosomes were visualized by gold-labeled LDL complexes. The binding, internalization, and degradation of LDL by subconfluent, non-contact-inhibited endothelial cells was quantified using 125I-labeled LDL. The LDL metabolism of endothelial cells derived from diabetic animals was increased by about 40% compared to endothelial cells derived from nondiabetic animals.

Animals↗
PubMed1985

Tangier disease: a disorder of intracellular membrane traffic.

The interaction of human high density lipoproteins (HDL) with isolated human monocytes from control and Tangier patients was studied in tissue culture experiments. It was observed that normal monocytes, similar to mouse peritoneal macrophages, bind HDL to a cell surface receptor, internalize the bound HDL particles, transport the internalized HDL intracellularly through the cytoplasmic compartment without significant degradation, and ultimately resecrete intact HDL. The cellular interaction of Tangier monocytes with normal HDL was markedly different from control monocytes. HDL binding to Tangier monocytes was moderately increased and cell-associated HDL radioactivity was 6- to 10-fold enhanced in Tangier monocytes. The bulk of the internalized HDL, however, was detected in secondary lysosomes. Only minor amounts of the internalized HDL were resecreted from the Tangier monocytes, and most of this was degraded. These data suggest that the cellular metabolism of HDL is abnormal in Tangier monocytes. It is postulated that Tangier disease may be a disorder of intracellular membrane traffic in which HDL is diverted into the lysosomal compartment and degraded instead of being secreted through its regular transcellular route.

Adolescent↗
PubMed1985

Receptor domains in the plasma membrane of cultured mouse peritoneal macrophages.

Using the platinum-carbon surface replication technique, the distribution of receptors for two colloidal gold labeled lipoproteins (acetylated low density lipoprotein and high density lipoprotein), iron-saturated transferrin and bovine serum albumin in the plasma membrane of cultured mouse peritoneal macrophages was mapped. The plasma membrane surface of cultured peritoneal macrophages exhibits three morphologically distinct regions which have shown differences in the distribution, density and dynamics of these receptors. The flat border of the plasma membrane surfaces are the domains that appear to have the highest concentration of transferrin receptors, whereas the intermediate regions contain most of the receptors for acetylated low density lipoprotein and high density lipoprotein. Bovine serum albumin receptors are concentrated at the edge of the cells and had a uniform distribution on the rest of the cell surface. The functional significance and the mechanisms by which these regional differences in the distribution of the receptors are generated and then maintained are not yet known.

Animals↗
PubMed1985

Demonstration of receptor binding of two apo-B containing lipoproteins by differential labelling with colloidal gold.

The interaction of two types of apo-B containing lipoproteins, human low density lipoprotein (LDL) and lipoprotein-a (Lp(a], with cultured human skin fibroblasts was studied by differential colloidal gold labelling in conjunction with thin sectioning and surface replication techniques. After separate exposure of the fibroblasts to either gold labelled LDL or Lp(a) for 15 to 30 min at 37 degrees C, labelled lipoproteins were predominantly found in coated pit areas. Excess of unlabelled LDL or Lp(a) completely displaced the gold labelled lipoproteins, indicating specific binding by the LDL-receptor. Simultaneous exposure of fibroblasts to LDL-16 nm gold and Lp(a)-40 nm gold conjugates revealed that both LDL and Lp(a) are bound in the same coated pit and internalized into the same endosome. In contrast to native lipoproteins, gold labelled acetylated lipoproteins were found diffusely distributed on membrane surface areas predominantly representing fibronectin-containing fibrils.

Apolipoproteins B↗
PubMed1985

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. II. Binding sites of proteoglycans on the cell surface.

Exogenous proteoglycans stained for electron microscopy with colloidal gold and/or cuprolinic blue bind to the surface of cultured arterial smooth muscle cells at two different sites. (I) About 20% of the proteoglycans adsorbed to the cells from the culture medium interact as monomeric and multimeric proteoglycans with smooth or coated membrane areas. (II) The bulk of exogenous proteoglycans exhibits high affinity binding to cell membrane-associated 10 nm fibrils containing or being closely associated with fibronectin and to collagen. It is suggested that the self association of proteoglycans and their binding to the cell membrane and to cell surface-associated fibronectin and collagen are important for maintaining an appropriate micro-environment for the cultured cells.

Arteries↗
PubMed1984

Double labeling of lipoprotein receptors in fibroblast cell surface replicas.

Using gold particles of distinctly different sizes as markers, the simultaneous visualization of two types of lipoprotein binding sites in surface replicas of the human fibroblast plasma membrane was achieved. Double-labeling experiments based on this principle have been carried out using sequential or simultaneous exposures of fibroblast cultures to a variety of lipoprotein species. Examples of the results obtained are illustrated, and the application of this approach to the analysis of the distribution, the relationship between, and the specificity of different classes of receptor is discussed.

Cell Membrane↗
PubMed1984

Topography and dynamics of receptors for acetylated and malondialdehyde-modified low-density lipoprotein in the plasma membrane of mouse peritoneal macrophages as visualized by colloidal gold in conjunction with surface replicas.

The topography and dynamics of receptors for acetylated (acetyl) and malondialdehyde-modified (MDA) low-density lipoprotein (LDL) in the plasma membrane of cultured mouse peritoneal macrophages were investigated using a new technique. Modified LDL labeled with gold particles was used to visualize LDL receptors in the plane of the plasma membrane in platinum-carbon surface replicas of critical point-dried cells. It was found that the native distribution of unoccupied acetyl-LDL receptors is diffuse, whereas unoccupied MDA-LDL receptors are preclustered in the plasma membrane. Competition and double labeling experiments suggest the existence of two distinct classes of receptor sites for acetyl-LDL and MDA-LDL.

Acetylation↗
PubMed1984

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. I. Endocytosis and intracellular translocation of proteoglycan-gold conjugates.

Proteoglycans (Mr approximately 200 000) isolated from bovine arterial tissue were decorated with 17 nm diameter gold particles for tracing in electron microscopic thin sections and surface replicas. Lysine and arginine residues of their proteoglycan protein core are assumed to be essential for gold conjugation. The resulting proteoglycan-gold conjugates, which appear as pearl string-like gold strands of about 170 nm in length were used to visualize binding, endocytosis and intracellular translocation of proteoglycans by homologous cultured arterial smooth muscle cells. The proteoglycan-gold conjugates bind to coated as well as to non-coated cell surface membrane areas at 4 degrees C. This is followed by the formation of membrane invaginations. Postincubation at 37 degrees C leads to a time-dependent uptake of proteoglycan-gold conjugates via non-coated and coated vesicles which after fusion are translocated to multivesicular bodies and to large sized vesicles within 1 h. After conversion of these vesicles to lysosomal compartments the gold particles are uncoupled from the proteoglycans and are concentrated within residual vacuoles. From these vacuoles the gold particles are extruded. In contrast to the surface-bound proteoglycan-gold conjugates the released gold particles are condensed to bulky aggregates. The results, which include competition, inhibition and pulse chase experiments, extend biochemical data on endocytosis and degradation of proteoglycans.

Animals↗
PubMed1983

Freeze-fracture studies on tardigrade cuticle.

The cuticles of the heterotardigrade Echiniscus testudo and the eutardigrades Macrobiotus hufelandi and Milnesium tardigradum have been studied using freeze-fracture technique. Most of the layers seen in conventional TEM micrographs can be visualized. There is no clear evidence that the trilaminar components of the cuticle such as the outer epicuticle and the tripartite layer separating epi- and intracuticle or procuticle (whose membranous origin has been suggested by previous authors) fracture like a lipid bilayer. Microfibres not resolved or only poorly resolved by TEM can be recognized in the procuticle of all three species. Obviously their visualization depends upon the fracture angle. In Echiniscus testudo and Milnesium tardigradum the intracuticle or at least parts of it show a wavy arrangement of microfibres. Parts of the ventral intracuticle of E. testudo fracture in an obviously non-random pattern revealing distinct sublayers.

Animals↗
PubMed1983

Variability of the topography of low-density lipoprotein (LDL) receptors in the plasma membrane of cultured human skin fibroblasts as revealed by gold-LDL conjugates in conjunction with the surface replication technique.

In this investigation the membrane-perturbing effect of filipin, a polyene antibiotic which reacts specifically with cholesterol or cholesterol-like compounds in cell membranes, has been exploited to study the distribution of coated pits in cultured human skin fibroblasts. The coated pits, showing no filipin-cholesterol complexes, occurred singly or in clusters without apparent localization of either type to a particular region of the fibroblast membrane. Colloidal gold, conjugated to low-density lipoprotein, has proven to be an excellent marker, allowing the localization of low-density lipoprotein receptors on the surface of cultured cells. A pattern similar to that for the coated pits in the plasma membrane fracture faces was observed in the distribution of gold-low-density lipoprotein conjugates in surface replicas, indicating that the low-density lipoprotein receptors are associated with these coated pits. It was shown that there is an apparent heterogeneity in the distribution of low-density lipoprotein receptors, from cell to cell and even among different areas of the same cell membrane. The binding capacity for gold-low-density lipoprotein complexes, as represented by the extent of surface labeling, was directly related to the cell variety within the culture or to the particular experimental procedure. The observation of differences in the distribution of gold-low-density lipoprotein conjugates, even among adjacent coated pits, provides evidence for various stages of activity of the low-density lipoprotein receptors corresponding to incorporation, mobility, and internalization.

Cells, Cultured↗
PubMed1983

Kidney tubule cell injury of male rats after estrogen treatment.

Ultrastructural changes in the tubular epithelium of the rat kidney following a large dose of estrogen (300 micrograms per week for 20 weeks) were studied by means of electron microscopy. The ultrastructural changes in estrogen-treated rats were confined to the proximal tubule. The changes consisted of intracytoplasmic vacuoles and dilatation of the intracellular space between adjacent tubule cells. The cisternae of the endoplasmic reticulum exhibited some degree of vesiculation characterized by club-like formations. In addition, large masses of collagen could be observed within the peritubular capillaries. In contrast to the controls the tubular cells of estrogen-treated rats exhibited large areas or granules with dense inclusions and membranous, filamentous material in a process of being transformed into an autolysosome and residual body. The results are discussed in the light of those of other authors.

Animals↗