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R Dermietzel

Publications and source records attributed to R Dermietzel.

At least 55 records · Page 3Linked to original sources
PubMed1989

cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.

Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal calf serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed approximately 8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (alpha-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. alpha-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect on mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intercellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Immunochemical and immunocytochemical characterization of a novel monoclonal antibody recognizing a 140 kDa protein in cerebral pericytes of the rat.

A monoclonal antibody that recognizes a 140 kDa peripheral plasma membrane protein in pericytes of nervous tissues of the rat is described. Microvessels of brain cortex and perineurium of peripheral nerves are shown to react positively to this antibody. The antigen is absent in brain regions that lack a blood-brain barrier, i.e., choroid plexuses and area postrema. Antigen expression starts as early as day 18 of embryonic development. By means of immunoelectron microscopy the 140 kDa antigen was detected as clusters along the entire circumference of cerebral pericytes. The same antigenic determinant is also expressed in apical domains of plasma membranes of a variety of transporting epithelia, such as hepatocytes, enterocytes of the small intestine, and epithelial cells of proximal tubules of the kidney. We postulate the 140 kDa protein as being a constituent of the pericytes involved in regulative functions of the blood-brain barrier.

Animals↗
PubMed1988

Gap junction formation in rabbit uterine epithelium in response to embryo recognition.

Gap junction formation was studied in the uterine epithelium of nonpregnant, pregnant, and pseudopregnant rabbits in the periimplantation phase (6, 7, 8 days post coitum/post human gonadotropin injection) using freeze-fracture and immunocytochemistry as well as intracellular Lucifer yellow injection. At implantation (7 days post coitum) the uterine epithelial cells of the implantation chamber become junctionally coupled as evidenced by all three methods used. Gap junction protein (26K) becomes detectable immunocytochemically with a monoclonal antibody at 6 days post coitum in the epithelium surrounding the blastocyst, i.e., in the forming implantation chamber. The same sequence of events, starting with the presence of the gap junction protein before cell-to-cell coupling becomes evident, was observed in the blastocyst-free segments 1 day later. In contrast, uterine epithelium of nonpregnant and pseudopregnant animals in comparable phases shows an extremely low degree of coupling. The presence of the blastocyst is a necessary condition for the induction of gap junctions as demonstrated by unilateral pregnancy produced by tubal ligation. Thus, gap junction formation is one of the first maternal responses to a locally acting signal of the blastocyst.

Animals↗
PubMed1988

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.

Actinin↗
PubMed1987

Major loss of the 28-kD protein of gap junction in proliferating hepatocytes.

There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.

Animals↗
PubMed1987

Simultaneous light and electron microscopic observation of immunolabeled liver 27 KD gap junction protein on ultra-thin cryosections.

We report on immunolabeling of gap junction protein in rat liver. Simultaneous light and electron microscopic immunolabeling of ultra-thin frozen sections was performed to confirm that the antigenic targets of polyclonal antibodies and a monoclonal 27 KD antibody (12/1 C5) are the gap junctions. Our results clearly demonstrate that the immunoreactive sites determined by indirect immunofluorescence correspond to immunogold-labeled gap junctions identified in the same section according to electron microscopic criteria. Our results also support the concept that the 27 KD protein is a major constituent of gap junctions.

Animals↗
PubMed1986

Reduced number of gap junctions in rat hepatocarcinomas detected by monoclonal antibody.

A new rat monoclonal antibody was characterized which recognized the 26K protein in gap junctions of mouse, rat and human liver as shown by immunoblot, indirect immunofluorescence, and immunogold electron microscopy. This monoclonal antibody was used to investigate the abundance of gap junctions in chemically induced rat hepatocarcinomas. In comparison with livers of control animals we found in hepatocarcinomas an average decrease of 71% in the number of gap junctional immunofluorescent spots. A corresponding decrease of the total amount of the 26 K protein was detected by quantitative immunoblot. Changes in the proliferative state as well as in intercellular adhesion of hepatocarcinoma cells in contrast to normal hepatocytes might have contributed to cause this decrease of gap junctions in tumor tissue. Possibly the partial loss of gap junctions provided a selective advantage for those preneoplastic liver cells which developed into rapidly proliferating tumor cells.

Animals↗
PubMed1986

Carbonic anhydrase in the sarcoplasmic reticulum of rabbit skeletal muscle.

Sarcoplasmic reticulum vesicles and mitochondria were prepared from red and white skeletal muscles of the rabbit. The preparations were characterized in terms of their specific activities of citrate synthase, basal (Mg2+-dependent) and Ca2+-dependent ATPase (the latter two in the presence of NaN3 and ouabain), and their specific carbonic anhydrase activities were determined. Skeletal muscle mitochondria had high specific activities of citrate synthase (700-1200 mu. mg protein-1) and low carbonic anhydrase activities (0.1-0.4 u. ml mg protein-1). The latter are likely to be due to a contamination of the preparations with sarcoplasmic reticulum (s.r.) Preparations of s.r. vesicles showed negligible activities of citrate synthase and the expected differing patterns of basal and Ca2+-dependent ATPase in red and white muscles. Specific carbonic anhydrase activities in s.r. from both muscle types were high (2-4 u. ml mg protein-1). The highest carbonic anhydrase activity, 11 u. ml mg protein-1, was found in s.r. from rabbit m. masseter. The inhibition constant of s.r. carbonic anhydrase towards acetazolamide was 4-6 X 10(-8) M and similar but not identical to that of cytosolic carbonic anhydrase II. It appears possible that the carbonic anhydrase II-like enzyme previously found by us in muscle homogenates (Siffert & Gros, 1982) originates from the s.r. Histochemical studies using the dansylsuphonamide method described previously (Dermietzel, Leibstein, Siffert, Zamboglou & Gros, 1985) showed an intracellular pattern of carbonic anhydrase staining compatible with the presence of the enzyme in s.r.: spots homogeneously distributed across the fibre cross-sections in transversely sectioned fibres and thin, longitudinally oriented, bands in longitudinally sectioned fibres. It is estimated that s.r. carbonic anhydrase accelerates CO2 hydration within the s.r. approximately 1000-fold. Thus, CO2 and HCO3- react fast enough to provide a rapid source and sink for protons leaving and entering the s.r. in exchange for Ca2+.

Acetazolamide↗
PubMed1985

Extravasation of polymorphonuclear leukocytes from the cerebral microvasculature. Inflammatory response induced by alpha-bungarotoxin.

Postcapillary venules represent the segment of the microvasculature most vulnerable to inflammatory processes. While there is a considerable body of data on the peripheral vasculature, little is known about the primary events occurring during inflammatory reactions in cerebral blood vessels. We introduce here a model by which the migration of polymorphonuclear leukocytes through the CNS endothelial barrier can be studied. Alpha-bungarotoxin is used as a chemotactic agent and is shown, for the first time, to act by activating the complement cascade. Leukocytes migrate through the endothelium transcellularly. Two modes of migration are described: a direct mode whereby the cells use temporary pores in the vessel wall as portals, and an indirect mode whereby the leukocytes leave the vascular compartment after being enveloped by and incorporated into endothelial cells. The functional implications of these findings lead us to conclude that the direct mode of migration is a causal agent in the massive breakdown of the blood-brain barrier under acute inflammatory conditions.

Animals↗
PubMed1985

Mapping of cholinoceptive(nicotinoceptive)neurons in the lower brainstem: with special reference to the ventral surface of the medulla.

The distribution of cholinoceptive neurons in the lower brainstem of the rat was investigated by means of a histochemical method for specific acetylcholinesterase. Nicotinoceptive neurons were characterized using an alpha-bungarotoxin-horseradish peroxidase conjugate for the detection of nicotinic acetylcholine receptors. For the first time a nearly complete mapping of the location of cholinoceptive (nicotinoceptive) neurons of the lower brainstem was achieved. Special attention was focused on the organization of the cholinoceptive neuronal matrix of the ventral surface of the medulla, where regulative centers for vasomotor and respiratory control are located.

Acetylcholinesterase↗
PubMed1985

Mapping of different neuropeptides in the lower brainstem of the rat: with special reference to the ventral surface.

A neuropeptide map of beta-endorphin-, vasoactive intestinal peptide-, substance P-, and somatostatin-like reactive neurons and nerve fibers was made by means of immunohistochemistry. Indirect immunofluorescence was carried out in parallel to peroxidase-antiperoxidase reaction using a modified fixation technique. Special interest was directed to the superficial ventral regions of the medulla oblongata where regulative centers for respiration and circulation have been localized. The atlas presented offers a reliable tool for a precise neuromorphological localization of these neuropeptides in pharmacophysiological experiments.

Animals↗
PubMed1985

A fast screening method for histochemical localization of carbonic anhydrase. Application to kidney, skeletal muscle, and thrombocytes.

A simple method for histochemical localization of carbonic anhydrase using 5-dimethyl-amino-naphthalene-1-sulfonamide (DNSA) is described. Cryosections of tissues, or cell smears, are incubated in 3 to 10 X 10(-5) M DNSA and viewed in a fluorescence microscope. Upon excitation with ultraviolet light, sites of carbonic anhydrase localization can be identified by an intense blue fluorescence, which is due to the emission of blue light (lambda max = 470 nm) by carbonic anhydrase-DNSA complexes. This fluorescence can be largely suppressed by simultaneous incubation with 1 X 10(-4) to 2 X 10(-3) M concentrations of nonfluorescent carbonic anhydrase inhibitors, displacing DNSA from its binding site on the enzyme. Application of the method to kidney, skeletal muscle, and thrombocytes yields patterns of carbonic anhydrase localization that are in good agreement with results that have been obtained with a variety of other techniques.

Animals↗
PubMed1984

Gap junctions in several tissues share antigenic determinants with liver gap junctions.

Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labelling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded.

Animals↗
PubMed1984

A method for the preparation of cerebral blood vessels.

Elements of the cerebral vascular system of the rat have been prepared by simultaneous perfusion with fibrinogen and thrombin. The resulting fibrin fills and serves to support the vessels which can then easily be excised from the brain and prepared for light and electron microscopy. The structure and ultrastructure of cerebral blood vessels is well preserved by this technique.

Animals↗
PubMed1984

Cytoplasmic and cell surface structure of purified liver gap junctions revealed by freeze-drying.

After freeze-drying of purified liver gap junction plaques and vesicles the structural features of the inner and outer aspects of purified gap junctions were investigated. No structural details were seen on the cytoplasmic side of the connexions whereas on the cell surface side the connexions were organized in a paracrystalline pattern and exhibited a central depression or pore. We conclude that the central pore through each connexion varies in diameter along its length and that the closing site is located near the cytoplasmic face.

Animals↗
PubMed1983

Cytochemical demonstration of negative surface charges in central myelin.

Homogenates of central myelin were treated with ferritin derivatives having different isoelectric points. It was found that considerable amounts of cationic ferritin (pI 8.5-9.5) had access to the extracellular space, but that anionic ferritin (pI 4.0) and native ferritin (pI 4.5) did not. The electrostatic nature of the binding of cationic ferritin was demonstrated by treating the homogenates with poly-L-lysine and 1 M NaCl:both reagents led to a complete displacement of the bound cationic ferritin. Neither extensive trypsination nor neuraminidase treatment showed a significant effect on the intralamellar distribution of the bound cationic ferritin molecules. This suggests that the net negative charge on the extracellular myelin face stems primarily from acidic lipid groups in the membrane.

Animals↗