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H Robenek

Publications and source records attributed to H Robenek.

144 records · Page 8Linked to original sources
PubMed1980

Ultrastructural morphometric investigations on rat liver of young and adult rats after treatment with technical pentachlorophenol (PCP).

Age-dependent effects of technical pentachlorophenol (PCP) on male rat livers were investigated after a 15 day treatment with PCP, 30 mg/kg/d body weight. The liver tissues were investigated morphometrically at light and electron microscopical levels. Statistically significant alterations of nuclei and organelles of the hepatocytes were described.

Adipose Tissue↗
PubMed1980

Thin section and freeze-fracture studies of hexachlorophene induced alterations in the rat liver with special regard to intercellular junctions.

The ultrastructural changes in the liver cells of male Wistar rats induced by the oral and intraperitoneal application of hexachlorophene (HCP) were studied by using the freeze-fracture technique and by producing ultrathin sections. The rats were given the HCP once at a dose of 30 mg/kg body weight and were sacrificed 60 hours after treatment. The enlargement of bile canaliculi, the disorganization of tight junctions and the increase in number of gap junctions were the most prominent alterations observed in the liver cells. Furthermore, an increase in the smooth and rough endoplasmic reticulum and the microbodies were found. Pathological alterations in the mitochondria were also seen. In general, the effects of the intraperitoneal application on the liver were more pronounced than the effects of the oral application.

Animals↗
PubMed1980

A correlated thin section and freeze-fracture study of o-phenylphenol-indued alterations in the rat liver.

The ultrastructural changes in the liver cells of male Wistar rats induced by oral and subcutaneous application of o-phenylphenol were studied electron microscopically using the thin section and freeze-fracture technique. The rats were given the o-phenylphenol once at a dose of 2,500 mg/kg b.w. and were sacrificed 60 h after treatment. In the orally treated animals, alterations in the nuclei and nucleoli of the hepatocytes were the most prominent alterations. Furthermore, an increase in smooth endoplasmic reticulum, lysosomes and microbodies were found. After the subcutaneous injection of o-phenylphenol enlargements of the bile canaliculi, intercellular space and pathological alterations in the mitochondria were seen. A proliferation of the smooth endoplasmic reticulum, an increase of microbodies and a decrease in the rough endoplasmic reticulum could be found. Both groups showed an increase in small intracellular fat droplets in the hepatocytes. In general, the effects of the subcutaneous application on the liver were more pronounced than the effects of the oral application. Freeze-fracture replicas exhibited a disorganization of the zonulae occludentes and an apparent increase in the number and size of gap junctions. These alterations were interpreted as an attempt of the liver cells to counteract the intracanalicular pressure and to increase the mechanical stability of the liver tissue.

Animals↗
PubMed1980

The morphologic characteristics of intercellular junctions between normal human liver cells and cells from patients with extrahepatic cholestasis.

In freeze-fracture replicas the bile canaliculi of normal human livers showed a lumen of rather constant size with parallel margins. The zonula occludens consists of a complex anastomosing network of intramembranous ridges on the P face and complementary grooves of the E face of the plasmalemma of liver parenchymal cells. The zonula occludens is usually composed of three to five ridges running parallel to the lumen of the bile canaliculus that are surrounded by a looser meshwork of variable orientation. All tight junctions observed in control replicas appeared as continuous barriers without any disruptions. Extrahepatic cholestasis produced considerable morphologic alterations in the canaliculi and tight junctions. The lumen of the canliculi enlarged, and the microvilli disappeared. Side branches, irregularities, and outpouchings of the canalicular membrane extending into the cytoplasm of the hepatocytes were frequently observed. The complexity of the branching pattern and the number of strands in the zonulae occludentes changed extensively. Junctional strands away from their usual pericanalicular location were present on the lateral surface of the plasma membrane. The altered zonulae occludents contain regions in which the strands had a fragmented appearance or were completely absent. These discontinuities in the junctional meshwork provide a direct pathway between the lumen of the bile canaliculus and the intercellular space. They strongly suggest a leakage of the canaliculosinusoidal barrier. Of further interest is the diffuse aggregation of the usually randomly distributed intramembranous particles of the P face of the plasmalemma. The aggregates consist of 10-50 individual particles. Concomitantly, the desmosomes appeared to be more numerous than normally. The number and structure of gap junctions remained unaffected. The results of this investigation are discussed in relation to those obtained after experimental bile duct ligation in rats.

Adult↗
PubMed1979

Carbon tetrachloride induced proliferation of tight junctions in the rat liver as revealed by freeze-fracturing.

Application of carbon tetrachloride produced a progressive proliferation of tight junctions in the rat liver. This system proved to be rapid and highly reproducable and affords the opportunity for tracing the fate of tight junctions in freeze-fracture replicas, facilitating investigations on their formation and function. Beginning on day one carbon tetrachloride treatments resulted in the progressive loosening and fragmentation of the junctional meshwork. After three to four days the membrane outside the zonulae occludentes was extensively filled with proliferated discrete junctional elements often forming complex configurations. From the fifth day on the zonulae occludentes were restricted again predominantly around the bile canaliculus margins. But the junctional meshwork of the zonulae occludentes remained loosened in comparison to those in the control rats. It could be shown that tight junction proliferation on the lateral surface of the plasmalemma occurred both through de novo formation from discrete centers of growth by addition of intramembranous particles and through reorganization of preexistent junctional strands of the fragmented zonulae occludentes bodies. Whereas the large gap junctions close associated with the zonulae occludentes remained more or less unaffected during the experiments, small gap junctions increased in number after five days and were located at the margin or in the tight junction domain. It is assumed that the degeneration of the tight junctions served as a pool for intramembranous particles which form the gap junctions. The results of these observations are discussed in relation to those obtained in other systems.

Animals↗
PubMedDate not supplied

Polyclonal antibodies against formaldehyde-modified apolipoprotein A-I. An approach to circumventing fixation-induced loss of antigenicity in immunocytochemistry.

A central requirement for the subcellular localization of synthesized and/or endocytozed lipoproteins is the availability of antibodies against the antigen to be localized. The preparation of cells for electron microscopy, in particular the fixation and embedding routine, influences the antigenicity, often resulting in a markedly reduced labeling intensity. To ameliorate fixation-induced changes in antigenicity, we produced antibodies against pre-fixed human apolipoprotein (apo) A-I. Purified apo A-I was fixed with 4% formaldehyde and was used to raise polyclonal antibodies in rabbits. The antiserum was purified by protein A-Sepharose followed by affinity chromatography with the fixed antigen coupled to vinylsulfone-activated agarose. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed lipoproteins. Nonspecific binding to unfixed or to fixed apolipoproteins was not observed. Thus, the antibodies reacted specifically with apo A-I and recognized the fixed as well as the unfixed protein. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, indicating that the antibodies were directed against epitopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells which are known to synthesize apo A-I. When HepG2 cells were exposed to anti-apo A-I antibodies followed by a secondary fluorescein isothiocyante-labeled antibody, fluorescence was found intracellularly in distinct regions. Electron microscopy revealed that the endoplasmic reticulum, and in particular the trans elements of the Golgi complexes, were the main compartments stained for apo A-I both in HepG2 cells, as shown by the immunoperoxidase technique, and in human hepatocytes, as shown by the protein A-gold technique on ultrathin cryosections. Furthermore, we were able to localize apo A-I in foam cells of the human aortic plaque by postembedding immunolabeling techniques. These findings demonstrate the potential of antibodies to fixed lipoproteins in impaired localization at the light microscopic and electron microscopic levels.

Animals↗
PubMedDate not supplied

Dysregulation of lipid metabolism in Tangier monocyte-derived macrophages.

The cellular defect in Tangier mononuclear phagocytes (MNP) was shown to be associated with significant abnormalities in cellular phospholipid, triglyceride, and cholesteryl ester metabolism by using various radiolabeled precursors (32Pi, 3H-serine, 3H-choline, 14C-acetate, and 14C-oleic acid). Tangier MNP expressed increased rates of synthesis for phospholipids (twofold), triglycerides (fivefold), and cholesteryl esters (threefold) as compared to normal MNP when incubated in McCoy's medium containing 0.2% human serum albumin. The turnover rate of cellular phospholipids was also enhanced, while the turnover rates for triglycerides and cholesteryl esters were normal, thus leading to the accumulation of a larger pool of labeled triglycerides and cholesteryl esters in Tangier MNP. The individual phospholipid classes, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine were similarly affected. Cholesterol loading led to approximately 30% down-regulation of phospholipid synthesis in normal cells, but Tangier MNP showed a smaller response. When nonloaded normal MNP were exposed to high density lipoprotein3 (HDL3), they diminished cellular cholesterol esterification mediated by acyl-CoA:cholesterol acyltransferase (ACAT); in Tangier MNP, ACAT activity increased in the presence of HDL3. When cholesterol-loaded normal and Tangier MNP were treated with HDL3, an up-regulation of phospholipid synthesis was observed in both cell types, but Tangier MNP showed a smaller response. We conclude that the defect in Tangier disease, which we recently described as a "disorder of intracellular traffic" (Schmitz et al. Proc Natl Acad Sci USA 1985;82:6305-6309), is associated with a dysregulation of cellular lipid metabolism, leading to an overproduction of triglycerides and esterified cholesterol and to enhanced synthesis and catabolism of phospholipids.

Adult↗
PubMedDate not supplied

Display of low density lipoprotein receptors is clustered, not dispersed, in fibroblast and hepatocyte plasma membranes.

Although the principal details of low density lipoprotein (LDL) uptake by receptor-mediated endocytosis and its subsequent intracellular fate have been thoroughly investigated, an aspect of this mechanism that continues to provoke controversy concerns the manner of display of LDL receptors upon their initial insertion at the cell surface. While our studies based on electron microscopy of platinum/carbon replicas of gold-labeled cells have previously suggested a clustered display pattern, others have concluded, before and since, that LDL receptors are inserted individually at random widely dispersed sites in the plasma membrane. In this article, we present a series of experiments designed to discriminate between these competing hypotheses. In addition to the use of LDL-colloidal gold complexes, visualized electron microscopically, on cells subjected to a variety of experimental procedures, these experiments include the application of anti-apolipoprotein B-100 antibodies, anti-LDL-receptor antibodies, and direct visualization of native (unlabeled) LDL molecules at the cell surface. All results point to a loose-cluster arrangement, not one involving widely dispersed individual units, as the initial display pattern of newly inserted LDL receptors. A comparison of LDL and beta-very low density lipoprotein receptor distribution in fibroblasts and hepatocytes suggests that this cluster pattern is a characteristic of the LDL (apolipoprotein B/E) receptor across cell types, but that the closely related apolipoprotein E receptor differs in that it is inserted individually in a highly dispersed state, in common with a variety of other receptor types.

Animals↗
PubMedDate not supplied

Abnormal processing of Golgi elements and lysosomes in Tangier disease.

It has been demonstrated that the cellular defect in Tangier disease is associated with morphological abnormalities in the lysosomal compartment and in the Golgi apparatus of mononuclear phagocytes (MNPs) after lipid loading by exposure to acetylated low density lipoproteins (acetyl-LDLs). On exposure to acetyl-LDL, Tangier MNPs accumulate two unusual types of vacuoles that were not observed in control MNPs. The electron-lucent type I vacuoles are round or ovoid, form aggregates, and are filled with fine flocculent or fibrillar material. Type II vacuoles are filled with more electron-dense material, are larger, and contain scrolls and lamellae with varying degrees of electron opacity. By immunoelectron microscopy both types of vacuoles could be identified as lysosomes. This abnormality of the lysosomal compartment in Tangier MNPs was not observed in Tangier fibroblasts, where the few lysosomes detected were found to be similar to those of controls. Morphological analysis of the Golgi apparatus in Tangier MNPs revealed numerous dilated Golgi cisternae, which were distributed more widely throughout the cell. However, both Tangier MNPs and especially Tangier fibroblasts showed a marked hyperplasia of the Golgi complex. Specific staining of the Golgi apparatus with the fluorescent ceramide analogue N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproylsphingosine (C6-NBD-ceramide) showed that Tangier fibroblasts incorporated significantly higher amounts of C6-NBD-ceramide than did control fibroblasts. Moreover, the Tangier fibroblasts that appear generally larger than normal fibroblasts also show significant staining along the cytoskeleton, which may indicate the association of fluorescent ceramide and sphingomyelin to cytoskeletal elements. Phospholipid synthesis and catabolism were studied in Tangier fibroblasts from five patients with the use of tritiated choline as a tracer. A significantly higher incorporation rate was measured for sphingomyelin (176 +/- 18% of control) and phosphatidylcholine (144 +/- 12% of control). A moderate increase in catabolism was found for sphingomyelin (20 +/- 8% of control) and phosphatidylcholine (11 +/- 4% of control). These data are principally in agreement with our former studies of the abnormalities of phospholipid metabolism in Tangier MNPs (G. Schmitz et al, Arteriosclerosis 1990; 10:1010-1019). It is concluded that the defect in Tangier disease that we have recently described as a "disorder of intracellular traffic" (G. Schmitz et al, Proc Natl Acad Sci U S A 1985;82:6305-6309) is also expressed in Tangier fibroblasts. Tangier disease seems to be associated with morphological abnormalities of intracellular organelles, thus strongly indicating that the translocation disorder in this disease affects the Golgi apparatus and the regular processing of lysosomes and shows abnormalities in cellular lipid metabolism.

4-Chloro-7-nitrobenzofurazan↗
PubMedDate not supplied

Collagen synthesis in cultured aortic smooth muscle cells. Modulation by collagen lattice culture, transforming growth factor-beta 1, and epidermal growth factor.

We investigated the effects of transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the protein synthesis and production of collagen in cultured smooth muscle cells (SMCs) from the aortic media of pigs. SMCs were cultured as monolayers on plastic as well as in three-dimensional collagen lattices to gain some information about the influence of a preexisting collagenous matrix on the growth factor-induced effects. A 48-hour exposure of SMCs to TGF-beta 1 at concentrations of 5 ng/ml in the presence of 1% serum caused a marked enhancement of the production of collagen and noncollagen proteins. The rate of net collagen production by SMCs exposed to TGF-beta 1 was approximately threefold higher than that of control cells. Moreover, TGF-beta 1 specifically stimulated collagen synthesis, resulting in a greater proportion of collagen in total proteins synthesized compared with controls. The preexisting matrix of collagen lattices affects the response of SMCs to TGF-beta 1 and EGF. In monolayer cultures the collagen proportion increased twofold under the influence of TGF-beta 1, whereas in collagen lattices the specific stimulation of collagen synthesis was lower. We found that EGF enhanced TGF-beta 1-induced protein production in collagen lattices but not in monolayer cultures. In addition, the protein production by SMCs was influenced differently by EGF in these culture systems. Taken together, these data show a mutual influence of growth factors and extracellular matrix components on collagen production in SMCs, thus indicating that TGF-beta 1 may be an important pathophysiological regulator of collagen metabolism in the vessel wall.

Animals↗
PubMedDate not supplied

Ca++ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. I. Characterization of cellular lipid metabolism.

The effects of the slow Ca++ channel blocker, nifedipine, and ACAT inhibitor, octimibate, on the cholesterol metabolism of cholesterol-loaded macrophages were compared. We demonstrated that apolipoprotein A-I containing high density lipoproteins (HDL) bind to specific receptor sites on macrophages, are internalized, take up cholesterol, and are then released from the cells as native lipoproteins. The ACAT inhibitor enhances HDL receptor activity and promotes HDL-mediated cholesterol efflux from cultured mouse peritoneal macrophages. In contrast, the Ca++ antagonist increases acetyl LDL-mediated cholesterol influx, abolishes the increase in HDL binding induced by cholesterol accumulation, enhances apo E synthesis, and promotes cholesterol efflux by a mechanism independent of the presence of HDL in the surrounding medium. Concomitantly, a decrease in nucleoside transporter activity, an increase in intracellular ATP hydrolysis, adenosine and cyclic AMP concentration, and a stimulation of the activities of acid and neutral cholesteryl ester hydrolase and ACAT indicated that protein kinase A-catalyzed phosphorylation reactions might be involved in the increase in cholesterol efflux. The Ca++ antagonist-induced efflux occurred only with lysosomal-associated cholesterol, while the ACAT inhibitor acted on the formation of cytoplasmic lipid droplets. The secreted lipoprotein particles contained 68% unesterified cholesterol and 21% phospholipids, 8% esterified cholesterol, and 3% triglycerides. The phospholipid components were: 72% phosphatidylcholine, 22% sphingomyelin, and 6% phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine. We conclude that macrophages release cholesterol in two ways: 1) an HDL-mediated release of unesterified cholesterol increasing upon ACAT inhibition, and 2) an HDL-independent secretion of cholesterol which can be amplified by Ca++ antagonists.

Acetylation↗
PubMedDate not supplied

Ca++ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. II. Characterization of intracellular morphologic changes.

The effects of the slow Ca++ channel blocker, nifedipine, and ACAT inhibitor, octimibate, on cholesterol-loaded macrophages were compared at the ultrastructural level. The Ca++ antagonist induced the formation of membrane-surrounded "lamellar bodies" originating from lysosomes. The macrophages secreted these lamellar bodies, which were rich in phospholipids and cholesterol, into the culture medium even in the absence of cholesterol acceptors. In contrast, the ACAT inhibitor induced the formation of lamellar bodies originating from lipid droplets, which were also surrounded by membranes. There is strong evidence that these latter membranes were newly synthesized at the margin of the lipid droplets by the endoplasmic reticulum. The lamellar bodies descending from lipid droplets after ACAT inhibitor treatment were not secreted by the cells. They were stored in the cytoplasmic compartment in the absence of high density lipoproteins (HDL). When HDL were added to the medium, the lamellar bodies specifically interacted with endosomes containing the internalized HDL particles and disappeared concomitantly with an enhanced HDL-mediated cholesterol efflux. From the biochemical data in the accompanying article and the morphological data here, we conclude that macrophages release cholesterol by two major pathways: 1) an HDL-independent secretion of lamellar bodies containing cholesterol, which originate from lysosomes, and 2) an HDL-dependent release of cholesterol via the formation of lamellar bodies descending from lipid droplets, which intracellularly fuse with HDL-containing endosomes. The cholesterol-enriched HDL are then secreted as intact lipoproteins.

Animals↗