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H Robenek

Publications and source records attributed to H Robenek.

At least 109 records · Page 6Linked to original sources
PubMed1983

Molecular forms of cathepsin D in coated vesicle preparations.

We have studied the polypeptide pattern of cathepsin D associated with coated vesicle fractions prepared from human placenta. In these fractions cathepsin D was about 35-fold enriched in the precursor polypeptides as compared to the unfractionated tissue extract. The enrichment was more prominent if the vesicles were fractionated in the presence of Triton X-100. Adsorption of exogenously added metabolically labelled cathepsin D precursor to the fractionated material was negligible. It is likely that the precursor and may be also the mature cathepsin D polypeptides are present in the matrix of the coated vesicles. This finding substantiates the idea that coated vesicles participate in the transport of newly synthesized cathepsin D into the lysosomes.

Cathepsin D↗
PubMed1983

Dynamics of low-density lipoprotein receptors in the plasma membrane of cultured human skin fibroblasts as visualized by colloidal gold in conjunction with surface replicas.

The aim of this study was to characterize further the human skin fibroblast membrane receptor for LDL. We herein report that LDL particles bound to colloidal gold in conjunction with the surface replication technique can be used to visualize steps in the movement of their receptors in the plane of the lipid bilayer and provide the first clear demonstration of the sequential clustering of LDL-receptors into coated pits. Competition experiments have shown that recycled LDL-receptors are inserted in plaques and not at random into unspecialized regions of the plasma membrane. The site of replacement of LDL-receptors into the plasma membrane corresponds to the site of formation of coated pits and their subsequent internalization.

Cell Membrane↗
PubMed1983

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. IV. Disintegration of bile canaliculi and disturbance of tight junction formation caused by vinblastine.

Treatment of cultured hepatocytes with vinblastine or colchicine caused striking perturbations of the structural organization of the biliary pole and of the junctional complexes. During the early hours of cultivation the reassociation of the bile canaliculi was impaired by the drug, whereas at later times in culture preformed canaliculi were disintegrated to small vesicular remnants lacking microvilli. Vinblastine did not impair tight junction formation per se. However, under the influence of the drug, tight junctional strands associated in an atypic manner perpendicular to the upper surface of the hepatocytes, whereas those strands lining the canaliculi were decomposed to smaller entities and dislocated within the lateral membrane. Concomitantly to the structural disintegration of the biliary pole an accumulation of vesicles in the pericanalicular cytoplasm was noted. As indicated by numerous filipin-induced lesions, they were characterized by a high content of membrane cholesterol. The apical pole and the contiguous membrane on the other hand contained only very few filipin-cholesterol lesions. These findings suggest that antimicrotubular drugs impair the fusion of pericanalicular vesicles with the luminal membranes of the canaliculi, thus interrupting the delivery of membraneous material to the apical pole. In addition, microtubules seem to play an important role in the coordinated development and the structural fixation of the biliary pole of cultured hepatocytes.

Animals↗
PubMed1982

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia, Urodela) using the antibiotic filipin.

The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipin-sterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.

Animals↗
PubMed1982

Alterations of tight and gap junctions in mouse hepatocytes following administration of colchicine.

Following the administration of colchicine at a dosage (1 mg/mouse) known to cause an antimicrotubular effect, membranes as well as tight and gap junctions of hepatocytes were studied using the thin-sectioning and freeze-fracturing technique. As early as 1 h after administration of colchicine the intercellular spaces were dilated and vacuoles were visible within the cytoplasm. The bile canaliculi became enlarged, and after lanthanum perfusion the tracer was found in the canalicular lumen, i.e., the tight junctions became permeable to the tracer. These findings correlated with a disorganized arrangement of the tight junctional strands of the zonula occludens. In some regions the strands showed interruptions and frequently ended freely in a diffuse pattern on the plasma membrane. Proliferation of tight junctions could be observed at various locations on the plasma membrane. The gap junctions also exhibited alterations. They showed an irregular outline with outpouchings, in addition to an enlargement in their total area from approximately 0.5 microns 2 in controls up to approximately 2 microns 2 in treated mice. The surface area occupied by these junctions increased from 4% (controls) to 20% (treated) of the hepatic plasma membrane. In the cytoplasm of hepatocytes from colchicine-treated mice gap-junctional vesicles were frequently observed. In view of the antimicrotubular effect of colchicine it is tentatively suggested that the intact microtubular system of the cell may play a decisive role in the regular formation of gap and tight junctions, either directly or indirectly via microfilaments.

Animals↗
PubMed1982

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse. II. A freeze-fracture study in conjunction with filipin.

The distribution, size, and configuration of intercellular junctions in the sebaceous tumor of mice were examined using the freeze-fracture technique. Three types of junctions were observed: desmosomes, gap, and tight junctions. Tight junctions in general consisted of short linear unbranched fibrils, and macular or complex tight junctional patterns were present only occasionally. Gap junctions ranged from small sports of 0.9 x 10(-3) micrometer2 to areas of approximately 0.46 micrometers2. Desmosomes were the most frequent junctional specializations, and it is concluded that they are at least partially responsible for this tumor not being metastatic. Filipin in conjunction with freeze-fracture showed filipin-sterol complexes on the plasma membrane, the nuclear envelope, and the membranes of the endoplasmic reticulum and mitochondria. The intercellular junctions were devoid of these complexes indicating that these regions are low in cholesterol.

Adenoma↗
PubMed1982

Changes in mouse hepatocytes caused by vincamin. A thin-sectioning and freeze-fracture study.

Mouse liver was examined following a single intraperitoneal application of 25 mg/kg body weight vincamine. The studies were made using the technique of thin-sectioning for electron microscopy as well as the freeze-fracture method. The thin-sectioning technique was useful for observing discrete changes such as an increase in the mitochondria and alterations in the bile canaliculi, e.g. a loss of microvilli and dilatation of the lumen. No other cytoplasmic changes could be observed. It was only with the aid of the freeze-fracture method that alterations in the cell contacts became visible. In contrast to the control animals the tight junctions in the liver tissue of mice treated with vincamin were disorganized and irregularly arranged. The gap junctions showed a very irregular contour as well. The question arises as to the extent to which the freeze-fracture method should be applied in the testing of pharmaceuticals, so as to exclude the possibility of damage to membranes.

Animals↗
PubMed1982

Effect of silybin on hepatic cell membranes after damage by polycyclic aromatic hydrocarbons (PAH).

Young female NMRI mice were given a single intraperitoneal injection of a PAH mixture containing 3 micrometers benzo(a)pyrene in a suspension of 0.5 ml tricapryline. 3 and 4 days later a number of the mice received injections of 100 mg/kg body weight of Silybin in the tail vein. Thin sections of the animals' livers were studied under the electron microscope using the freeze fracture technique. Following intraperitoneal injection of the PAH suspension, and without treatment with Silybin, marked dilatation of the intercellular space and frequent occurrence of intracytoplasmic vacuoles was observed in addition to pronounced alterations in the hepatocytic nuclei and mitochondria. Following the administration of Silybin these alterations were no longer observed, though 50% of the cell membranes exhibited a marked band-like proliferation of the tight junctions. This observation may be interpreted as supporting the postulated membrane-stabilizing effects of Silybin.

Animals↗
PubMed1982

Electron microscopic studies of the morphology of the corneal stroma in the rabbit cornea using the freeze-fracture technique.

The morphology of the corneal stroma in the rabbit cornea was investigated under the electron microscope using the freeze-fracture technique. The collagen fibrils were closely packed into collagen bundles. In the freeze-fracture replicas the amorphous ground substance could not be demonstrated. At the stroma membranes were shown with crater-shaped depressions on the P-face and corresponding elevations on the E-face. Regarding the topographical relationships between the membranes and other parts of the fibroblasts, it was concluded in this investigation that the crater-shaped structures probably constitute pores in the plasma membrane of fibroblasts.

Animals↗
PubMed1982

Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. I. Tight junctional analysis and tracer experiments.

Over a period of 20 weeks estradiol valerate (1.5 mg/kg body weight/week) was administered subcutaneously to male Wistar rats from which the livers were examined at four week intervals employing a freeze-fracture technique and colloidal lanthanum tracer studies. In connection with intrahepatic cholestasis, distinct alterations in the tight junctions were observed, consisting of disorganization, rarification and proliferation. Disruption of the tight junctions was not seen and colloidal lanthanum did not penetrate into the bile canalicular lumen. Holding the view that the term "leakiness" of tight junctions should be defined with reference to the tracer employed, we conclude that in the liver one tight junctional strand is sufficient to prevent the escape of larger bile constituents such as bile acids and that a back diffusion of bile acids over the tight junctional barrier does not play a role in the pathogenesis of the estrogen-induced cholestasis. Interruptions of tight junctions, as described by other authors, are interpreted as a secondary mechanical effect. On the other hand, we consider an increased permeability of the tight junctions to water and small solute molecules as probable; possibly this increased permeability is brought about by alterations in the microfilaments. A model for the pathogenesis of the estrogen-induced intrahepatic cholestasis is proposed.

Animals↗
PubMed1982

Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. II. Gap junctional analysis.

Treatment of male Wistar rats with estradiol valerate induced alterations in hepatic gap junctions as visualized by the freeze-fracture technique. The alterations involved the spacing, and regularity of packing of the membrane particles of the P face (PF) and complementary pits on the E face (EF), as well as internalization and changes in the number, size and shape of the junctional domains. In approximately 20% of the PF's of the lateral membrane of treated animals the nonjunctional IMPs were aggregated, while the bile canalicular membrane was never involved, maintaining its random distribution of particles. It is proposed that the changes in junctional area and the more general arrangement of the junctional particles may indicate a decrease in coupling between hepatocytes. The invaginations of gap junctions may represent a means for removing gap junctional membrane from the surface or may be an expression of a higher turnover of gap junctions. We assume that the alterations observed here are due to the specific effects of estrogen. This study addresses in detail a number of possible sites of activity and modes of action for estrogen.

Animals↗
PubMed1982

Freeze-fracture elucidation of hepatocyte membrane alterations following beta-pyridylcarbinol application.

The effect of beta-pyridylcarbinol on mice was investigated in long-term studies (21 days, daily dose of 0.1 mg) making use of thin-sectioning and freeze-fracture techniques. Thin sectioning merely revealed only subtle pathological changes including dilatation of the intercellular space and foundation of hepatocytic vacuoles. Only upon investigation using freeze-fracture was it possible to demonstrate more profound alterations, primarily involving the cell contacts, i.e. the gap and tight junctions. The organized structure of the tight junctions appeared dissipated. The meshwork was disorganized in some cases and reduced in size. Large, proliferative tight junctional maculae were frequently observed on the plasmalemma. In some cases the gap junctions had lost their round to oval shape and had increased in size to form large plaques protruding at numerous points. Moreover, even the bile canaliculi, which presented no pathological alterations on ultrathin section, exhibited damage as seen in freeze-fracture preparations. The lumen was uneven in contour and processes were visible extending into the adjacent cytoplasm. The present investigation shows that hepatocytic damage primarily affecting the membranes was first made evident using freeze-fracture technique. The issue is addressed as to the extent to which freeze-fracture technique should be employed in routine investigations of pharmaceuticals.

Animals↗
PubMed1982

A correlative study on the topographical distribution of the receptors for low density lipoprotein (LDL) conjugated to colloid gold in cultured human skin fibroblasts employing thin section, freeze-fracture, deep-etching, and surface replication techniques.

The topographical distribution of the receptors for low-density lipoprotein (LDL) conjugated to colloidal gold on cultured human skin fibroblasts was studied using a surface replication technique following critical point drying. In this correlative study, also employing thin sectioning, freeze-fracture and deep-etching techniques in conjunction with the polyene antibiotic filipin, it could be shown that LDL preferentially binds to certain microdomains of the plasma membrane, the so-called coated pits. The coated pits can be recognized after filipin treatment both on the P face and E face of the membrane, due to the fact that they are free of filipin-sterol complexes. Filipin was also of value in clearly delimiting the structure of nascent coated pits from their surroundings in surface replicas. Using filipin has the effect of increasing the contrast between the smooth surface of a coated pit and the rough surface of the remaining membrane caused by formation of filipin-sterol complexes. This study has shown that there are differences in the topographical distribution and number of gold particles from fibroblast to fibroblast and even between different areas of the same cell. The surface replication technique using goldlabelled LDL provides s suitable method for improving the interpretation of the spatial arrangement of the LDL receptors. The advantages of this technique are discussed in comparison with the methods previously employed to visualize LDL receptors.

Cell Membrane↗
PubMed1982

Cytochalasin B affects the gap and tight junctions of mouse hepatocytes in vivo.

This study investigates the effect of cytochalasin B at a dosage of 0.2 mg per mouse per day for a period of 7 days in an in vivo experiment on mouse liver. Using thin-sectioning and freeze-fracture technique both quantitative and qualitative analysis was made of membranes and cell contacts (gap and tight junctions). Significant alterations of both membranes and junctions were observed. The intercellular space showed vacuolar dilatation in some cases and there were vacuoles observed within the cytoplasm. The microvillar bile canaliculi were dilated. However, no colloidal tracer was observed within the lumen following lanthanum perfusion. With the aid of the freeze-fracture method it was possible to demonstrate that the strands of the tight junctions were highly disorganized. In some cases reduction and in other cases proliferation of tight junctions was observed. Large, proliferative plaques of tight junctions were found both in contact with the tight junctions of the bile canaliculus and ending freely on the plasmalemma. The gap junctions appeared enlarged as well. Their average size increased from 0.42 micron 2 to 0.90 micron 2 (p less than 0.005). The enlargement was also accompanied by an increase in the proportion of the plasma membrane occupied by the junctions: 3.42% in control animals, 10.25% in the livers of mice treated with cytochalasin B. Frequently evaginated and internalized gap junctions were seen in the experimental group. In view of the fact that cytochalasin B, in addition to other effects, also has an effect on the microfilament system of the cell, it may be supposed that microfilaments play a role in maintenance of the orderly structure or in the formation of tight and gap junctions. This remains hypothetical, however, and additional studies are necessary in order to conclusively clarify this issue.

Animals↗