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Biomedical subjects

R Gerhard

Publications and source records attributed to R Gerhard.

At least 19 recordsLinked to original sources
PubMed2006

[Antibiotic-associated diarrhea].

The incidence of antibiotic-associated diarrhea (AAD) differs with the antibiotic and varies from 15 - 25 %. Most cases of AAD are directly or indirectly caused by alterations of gut microflora by the antibiotics resulting in clinically mild AAD cases due to functional disturbances of intestinal carbohydrate or bile acid metabolism. Alternatively, changes in the gut flora allow pathogens to proliferate. Clostridium difficile is responsible for 10 - 15 % of all cases of AAD and almost of all cases of antibiotic-associated pseudomembraneous colitis. There is also a growing body of evidence which supports the responsibility of Klebsiella oxytoca for the development of antibiotic-associated hemorrhagic colitis. Diagnosing Clostridium difficile-associated diarrhea should be based both on fecal-cytotoxin detection and culture. With respect to specific therapy, metronidazol has become the first choice whereas treatment with oral vancomycin should be reserved for patients who have contraindications or intolerance to or who have failed to respond to metronidazole. Probiotics such as Sacharomyces boulardii can reduce the risk of development. Restrictive antibiotic policies (e. g. restricting clindamycin and cephalosporins) and the implementation of a comprehensive hospital infection control have also been shown to be effective in reducing the incidence of AAD.

Anti-Bacterial Agents↗
PubMed2004

Large clostridial cytotoxins.

The large clostridial cytotoxins are a family of structurally and functionally related exotoxins from Clostridium difficile (toxins A and B), C. sordellii (lethal and hemorrhagic toxin) and C. novyi (alpha-toxin). The exotoxins are major pathogenicity factors which in addition to their in vivo effects are cytotoxic to cultured cell lines causing reorganization of the cytoskeleton accompanied by morphological changes. The exotoxins are single-chain protein toxins, which are constructed of three domains: receptor-binding, translocation and catalytic domain. These domains reflect the self-mediated cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and execution of their cytotoxic activity by an inherent enzyme activity. Enzymatically, the toxins catalyze the transfer of a glucosyl moiety from UDP-glucose to the intracellular target proteins which are the Rho and Ras GTPases. The covalent attachment of the glucose moiety to a conserved threonine within the effector region of the GTPases renders the Rho-GTPases functionally inactive. Whereas the molecular mode of cytotoxic effects is fully understood, the mechanisms leading to inflammatory processes in the context of disease (e.g., antibiotic-associated pseudomembranous colitis caused by Clostridium difficile) are less clear.

Bacterial Toxins↗
PubMed2001

Electrolytic hydride generation atomic absorption spectrometry for the determination of antimony, arsenic, selenium, and tin--mechanistic aspects and figures of merit.

This article deals with the electrocatalytic and electrochemical mechanisms of hydride formation and their dependence on hydrogen overvoltage. A three-electrode-arrangement was used to determine the hydrogen overvoltage of different cathode materials (Pt, Au, Ag, glassy carbon, Cd, Pb, amalgamated Ag). The applicability of these cathode materials was tested for hydride formation using As(III), As(V), Sb(III), Sb(V), Se(IV), and Sn(IV). Glassy carbon is the most suitable cathode material for hydride generation with As(III), Sb(III), Se(IV), and Sn(IV). Hg-Ag is well suited for the production of stibine and arsine. As(III), As(V), Sb(III), and Sb(V) were all converted into their hydrides with efficiencies > 90%. A detection limit in the range of 0.11-0.13 microg L(-1) for As and Sb (sample volume 200 microL) was obtained for cathode materials with a high hydrogen overvoltage. The precision of replicate measurements was better than 5% calculated as variation coefficient. The accuracy of the presented method was verified by analysis of certified reference materials and tissues of cancer patients. The recovery rates for As and Se were calculated to be 93-108%.

Journal Article↗
PubMed2001

Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins.

The Rho GTPases, which belong to the Ras superfamily of low-molecular-mass GTP-binding proteins, are the preferred intracellular targets of bacterial protein toxins. The Rho GTPases RhoA/B/C, Rac1/2 and Cdc42 are the master regulators of the actin cytoskeleton. Clostridium difficile toxins A and B, the causative agents of the antibiotic-associated pseudomembranous colitis, are intracellularly acting cytotoxins which mono-glucosylate the Rho GTPases. Clostridium botulinum C3 toxin, which is not related to the clostridial neurotoxins, catalyses ADP-ribosylation of RhoA/B/C but not of other Rho GTPases. Glucosylation as well as ADP-ribosylation result in functional inactivation of Rho causing disassembly of the actin cytoskeleton.

Animals↗
PubMed2000

Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases.

Large clostridial cytotoxins catalyze the glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate. By site-directed mutagenesis of Clostridium sordellii lethal toxin and Clostridium difficile toxin B fragments, we identified tryptophan 102, which is located in a conserved region within the catalytic domain of all clostridial cytotoxins, to be crucial for UDP-glucose binding. Exchange of Trp-102 with alanine decreased the glucosyltransferase activity by about 1,000-fold and blocked cytotoxic activity after microinjection. Replacement of Trp-102 by tyrosine caused a 100-fold reduction in enzyme activity, indicating a partial compensation of the tryptophan function by tyrosine. Decrease in glucosyltransferase and glycohydrolase activity was caused predominantly by an increase in the K(m) for UDP-glucose of these mutants. The data indicate that the conserved tryptophan residue is implicated in the binding of the cosubstrate UDP-glucose by large clostridial cytotoxins. Data bank searches revealed different groups of proteins sharing the recently identified DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J., Jeckel, D., and Aktories, K. (1998) J. Biol. Chem. 273, 19566-19572) and a conserved region defined by a tryptophan residue equivalent to Trp-102 of C. sordellii lethal toxin. From our findings, we propose a novel family of glycosyltransferases which includes both prokaryotic and eukaryotic proteins.

Amino Acid Sequence↗
PubMed2000

Analysis of low-molecular-weight GTP-binding proteins in two functionally different intestinal epithelial cell lines.

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. In this study, for the first time, members of this family of proteins in two functionally different intestinal epithelial cell lines are identified and characterized. [alpha-32P]GTP blot overlay assays of cytosolic and membranous fractions revealed the presence of specific GTP-binding proteins in the range of 20-30 kDa (small GTPases) in both fractions, with considerably higher amounts in the membranous insoluble fraction. Analysis by two-dimensional electrophoresis, immunoprecipitation using monoclonal and sequence-specific polyclonal antibodies, and C3 exoenzyme-mediated ADP ribosylation demonstrated the presence of Ras, Rap, Rho, Rac, Rab, and several other small GTPases. The pattern of small GTP-binding proteins corresponded to the characteristics of the cell lines. Caco-2 cells showed a Rab5 protein that is known to be involved in endocytosis but was not found in T84 cells. On the contrary Rab3 has been shown to participate in secretory processes. It is highly expressed in T84 cells (sixfold compared to Caco-2 cells).

Caco-2 Cells↗
PubMed2000

Histomorphometrical study of the elastic fiber system in the anterior cerebral artery of man.

The aim of the present study was to quantify the distribution of the elastic fiber system within the wall of the anterior cerebral artery. The study is based on the works of Glynn (1940) and Stehbens (1989) concerning the incidence and origin of brain aneurysms and recent studies of the elastic fibers. The anterior cerebral artery was divided into three segments, S1, S2 and S3: S1 corresponds to the origin of the anterior cerebral artery, S2 is located at the junction of the anterior cerebral artery with the anterior communicating artery, and S3 at the junction of the rostrum and genu of the corpus callosum,which were submitted to routine histological procedures. A histomorphometrical study was undertaken using an estimation of the linear density (Ld) of the components of the fibrous elastic system which evaluates their full length in each segment. Data were analyzed using first order linear regression methods. The results show a decreasing quantity of elastic fibers in the three segments (S1>S2>S3). Study of the elastic fiber system may originate new concepts regarding the genesis of cerebral artery aneurysm.

Adult↗
PubMed1999

Involvement of K(+) channels in the relaxant effects of YC-1 in vascular smooth muscle.

This study addresses the question whether K(+) channels are involved in the vasorelaxant effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1 ). In rat aorta, guinea pig aorta, and guinea pig a. carotis, YC-1 inhibited contractions induced by phenylephrine (3 microM) more potently than those induced by K(+)(48 mM). In rat aorta, tetraethylammonium (10 mM), charybdotoxin (0.2 microM), and iberiotoxin (0.1 microM), but not glibenclamide (10 microM), attenuated the relaxant effects of YC-1. In guinea pig a. carotis, YC-1 (30 microM) induced a hyperpolarisation which was antagonised by 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 50 microM). In rat aorta, YC-1 (30 microM) increased the rate constant of 86Rb-efflux. The effect of YC-1 was potentiated by zaprinast (10 microM), but inhibited by ODQ (50 microM) or charybdotoxin (0.2 microM). In smooth muscle cells from rat aorta, YC-1 (10 microM) increased BK(Ca) channel activity. It is suggested that YC-1-induced vasorelaxation is partially mediated by the activation of K(+) channels.

Animals↗
PubMed1998

Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells.

The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers. Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B of Clostridium difficile. Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40%. Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation. Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively. Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation. In contrast, toxin B induced a 28-fold increase of permeation after 24 h. As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers. The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.

Bacterial Toxins↗
PubMed1995

Isolation and characterization of apical membrane vesicles of the rat distal colon.

In this study a method for the isolation of apical membrane vesicles of the rat distal colon was developed. It is based on the purification of intact membrane caps followed by separation of the vesiculated apical membranes on a discontinous sucrose gradient. Purification of the apical membrane vesicles revealed an 11-fold enrichment of the marker enzyme alkaline phosphatase compared with the homogenate, while marker enzymes of other subcellular structures showed negligible enrichments and recovery of activity. The membrane fluidity (lipid structural order) of the isolated membranes measured from the fluorescence anisotropy by several fluorophores also coincided with the typical structural order of apical membranes of the rat colon. Transport studies with the fluorescent dye acridine orange implied that a diffusion potential independent, amiloride-sensitive Na+ -H+ exchange mechanism is present in the isolated apical membranes. Furthermore, the results suggest that a possible short chain fatty acid (SCFA) absorption by simple passive diffusion of the undissociated form, preceded by intraluminal protonation of the SCFA anion, is not provided by this Na+ -H+ exchange transport in the luminal membrane of the absorptive cell.

Acid Phosphatase↗
PubMed1995

Characterization of putrescine transport across the intestinal epithelium: study using isolated brush border and basolateral membrane vesicles of the enterocyte.

Putrescine transport was investigated in isolated brush border and basolateral membrane vesicles prepared from the rabbit enterocyte. Brush border vesicles were oriented right-side-out and basolateral vesicles inside-out, forming a model representing uptake and extrusion across the intestinal epithelium. Putrescine transport across both membranes was initially rapid, and 66% of the equilibrium uptake was achieved within the first minute. According to osmoplots and measurements at 4 degrees C, 20% of total incorporation presented binding to the membrane. In order to estimate actual uptake into the vesicles, Km was calculated from the differences in putrescine incorporation at 37 degrees C and 4 degrees C, and was 12.7 mumol L-1 for brush border uptake and 38.2 mumol L-1 for basolateral extrusion. Putrescine uptake into brush border and basolateral membrane vesicles was not enhanced in the presence of an Na+ gradient. When Na+ was substituted with an uncharged solute, mannitol, putrescine incorporation was increased, indicating that putrescine uptake is not Na(+)-dependent and that cations might interfere with the carrier. Paraquat and methylglyoxalbis(guanylhydrazone), known to share the polyamine transport system, inhibited putrescine incorporation in both membrane vesicle preparations. Basolateral carrier showed significantly higher sensitivity to cations. We conclude that putrescine uptake across the apical membrane and extrusion across the basolateral membrane of the enterocyte are mediated by two different and independent carriers which differ in their electrical properties.

Animals↗
PubMed1995

Subcellular distribution of small GTP-binding proteins in the intestinal cell line Caco-2.

The present study describes the subcellular distribution of low molecular weight GTP-binding proteins in the human carcinoma cell line Caco-2. Highly enriched subcellular fractions of basolateral and brush border membranes were prepared by differential density centrifugation and divalent cation precipitation. Small molecular weight GTP-binding proteins were identified after SDS-PAGE transfer to nitrocellulose using [alpha 32P]-labelled GTP. Smg-proteins with molecular masses of 28, 27, 25 and 24 kDa were detectable in all fractions. Homogenate and brush border membrane fraction showed specific binding of [alpha 32P] GTP to proteins of a molecular mass of 21 kDa, while in the membrane fractions (apical, basolateral) a high enrichment of 24 kDa smg-proteins was detectable. Western blot analysis identified one of the 21 kDa proteins of the brush border membrane as rhoA. The homogenate of 4, 8, 11 and 14 days old Caco-2 cells showed different [alpha 32P]-GTP binding to 21, 27 and 28 kDa proteins. In conclusion, this study is the first showing the presence and asymmetrical distribution of smg-proteins among the various membrane components in the human carcinoma cell line Caco-2.

Caco-2 Cells↗
PubMed1994

Simultaneous preparation of rabbit intestinal brush border and basolateral membrane vesicles.

A highly reproducible method is described for simultaneous preparation of the rabbit intestinal BBMV and BLMV. When BLMV were purified on continuous sorbitol gradient the final enrichment was over ten-fold for both Na+, K(+)-ATPase and K(+)-activated phosphatase. BBMV were separated using repeated precipitation with magnesium and EGTA. Marker enzymes of the brush border membrane also showed over ten-fold enrichment. There was no contamination with other cellular organelles. The characteristics of the preparations were checked by SDS gel electrophoresis, which showed distinct protein patterns for both membranes; monoclonal antibodies for villin reacted with the BBMV but not with BLMV, showing the absence of contamination of the BLMV fraction. The quality of the membranes, assessed by D-glucose transport, showed good functional properties for both BBMV and BLMV. Orientation of the vesicles was checked by permeabilizing with SDS buffered with BSA and showed that BLMV are oriented inside out and BBMV right side out. The model simulates normal transepithelial intestinal transport at the level of membrane vesicles and could be applied in transport studies in physiology and pharmacology.

Animals↗
PubMed1982

[Therapy of hand joint fractures using the external fixator. Experiences - results].

In the treatment of multi-fragment fractures of the base of the radius plate osteosynthesis using the small-fragment instrument set is preferred to conservative treatment due to the superior results achieved. However, one still cannot dispense with an additional immobilization of the fracture in a plaster cast. The extension of intra-articular comminuted fractures of the base of the radius is a technically relatively simple procedure and does not require a supporting plaster cast. In this paper we report the results of the treatment of twenty-seven wrist-fracture patients who were treated by external fixator. We also describe the indications and operative techniques. The results are reported by functional and radiological findings. For a great number of cases the results were quite good. We can highly recommend this procedure in the treatment of wrist injuries due to the simplicity of the operative technique employed and minimal follow-up treatment needed in conjunction with the good results achieved.

Adult↗
PubMed1980

[Ender nailing - experiences and results (author's transl)].

The article reports on 69 patients with femoral fractures near the hip joint treated by means of Ender nails. The advantages of his method, which had already been reported earlier, are confirmed. Local complications occurring in our first follow-up group resulted in developing an operative technique which differs from the original method and is described in detail.

Aged↗