Search PubMed⌕ Search

Biomedical subjects

H Winkler

Publications and source records attributed to H Winkler.

At least 253 records · Page 14Linked to original sources
PubMed1985

Cell-free and cellular synthesis of chromogranin A and B of bovine adrenal medulla.

We have studied the cell-free and cellular synthesis of chromogranins A and B, two immunologically distinct protein families of adrenal chromaffin granules. Two cell-free systems (wheat germ and reticulocyte lysate) were used for translating messenger RNA isolated from bovine adrenal medulla. Two primary translation products could be immunoprecipitated in case of chromogranin A. In the presence of microsomes the two chromogranin A precursors (pre-chromogranins A) were converted into a single protein, apparently by the removal of different signal peptides. For chromogranin B only one precursor (pre-chromogranin B) was translated. In isolated chromaffin cells only one chromogranin A protein was synthesized which corresponded to the processed cell-free translation product. During prolonged incubation this protein became slightly larger and more acidic, probably due to glycosylation in the Golgi region. Chromogranin B is post-translationally converted to a significantly more acidic protein. It is concluded that proteolytic breakdown of newly synthesized chromogranin A and B in chromaffin granules is a slow process comparable to that of the enkephalin precursors. It is not yet known what function these chromogranins have and whether breakdown to smaller subunits is necessary for any function to evolve.

Adrenal Medulla↗
PubMed1985

Biogenesis of chromaffin granules: incorporation of sulfate into chromogranin B and into a proteoglycan.

The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse-labeled with [35S]sulfate. The radioactively labeled products were characterized by one- and two-dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein, much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000-100,000; pI 4.3-5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue-stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.

Adrenal Medulla↗
PubMed1985

[Toe transfer].

Today toe-to-hand transfer by micro-anastomoses is one of the most important methods in reconstructing severely injured hands. An experienced surgeon will obtain a survival rate of the transplant in about 94 to 96% of cases. All measures required can be performed by only one operation. The free toe-to-hand transfer guarantees the best functional and aesthetic results so far known.

Amputation, Traumatic↗
PubMed1985

[Significance of antithrombin III in clinical liver transplantations].

The activity of AT III was measured by chromogenous substrates in 18 of 28 patients before, between and after liver transplantation. In half of all cases basic pathological activities could be observed. During transplantation, AT III is on an average decreased to 43% of the initial level. Examples are demonstrated for important AT III deficiencies after liver transplantation in man. Long-time examinations have shown AT III to be in the normal range or higher in 1 to 60 months after transplantation when the graft function is correct.

Adult↗
PubMed1984

[The effect of enzymatic modification on lysinoalanine formation in field-bean protein isolate and beta-casein].

Lysinoalanine (LAL) was determined in alkali-treated partial hydrolysates (PH) of casein, peptides isolated from these PH and in PH of field-bean protein to clarify whether intermolecular or intramolecular LAL bridges are preferentially formed. Furthermore, the formation of LAS in plasteins was studied as a contribution to plastein research. The formation of LAL in the peptide mixtures of beta-casein and the decrease of the LAL content in the PH (as compared to intact proteins) indicates that the formation of LAL favours the intramolecular cross-linking of polypeptide chains. The LAL content decreases as the degree of hydrolysis of the PH of the field-bean protein isolate increases, and depends upon the protease used in the production of the hydrolysates. The LAL contents of the alkali-treated plasteins are less than those of the initial hydrolysates. The decrease of the LAL content is directly proportional to the hydrolysis proceeding during the plastein reaction.

Amino Acids↗
PubMed1984

[Incorporation of methionine into a peptic partial hydrolyzate from faba bean protein isolate by plastein reaction with thermitase].

The contents of free methionine methyl esters and methionine as well as the bound quantities of methionine were determined in order to evaluate the effectiveness of the covalent incorporation of methionine into peptic partial hydrolyzate in the course of the plastein reaction with addition of L-, DL- or D-methionine ethyl esters. The plastein reaction was performed with thermitase as reenzyme. The methionine incorporation was found to take place stereospecifically. The incorporation degree of the methionine ester is influenced by a thermitase-catalyzed and a non-enzymatic ester saponification.

Endopeptidases↗
PubMed1984

Erythrocyte pyruvate kinase deficiency: characterization of a new variant (PK "Aarau").

A new PK variant with moderate hemolytic anemia is described. The enzymes of the nonanemic parents show sigmoidal reaction kinetics, with normal kinetic parameters, but differ with respect to nucleotide specificity, thermostability, and the concentrations of the glycolytic intermediates in the erythrocytes. The most characteristic features of the patient's (daughter) enzyme are a 30% activity, hyperbolic reaction kinetics and only two bands in the SDS-gel electrophoresis instead of three bands observed with the parental enzymes. Moreover, the pH-optimum is shifted to the acidic range, the affinity for PEP and ADP is decreased, ATP inhibition is negligible and FDP-activation is roughly ten times smaller than with controls. The concentrations of 2,3-DPG, 2-PG and PEP in the erythrocyte are increased, but ATP decreased. As there is no consanguinity in the parents and their enzymes are different this PK mutant can be considered to be compound-heterozygous for two different mutant PK alleles.

Adenosine Triphosphate↗
PubMed1984

Chromogranin immunoreactivity in the central nervous system. Immunochemical characterisation, distribution and relationship to catecholamine and enkephalin pathways.

Chromogranin A, the major soluble protein of the chromaffin granules, was isolated from bovine adrenals and used for immunization of rabbits. Chromogranin (CHR) immunoreactivity was studied by immunochemical and immunohistochemical methods in the adrenal, pituitary, brain and spinal cord of cattle, sheep, rats and guinea pigs using two antisera neither of which cross-reacted with dopamine beta-hydroxylase. Detailed studies were done using tissues from sheep only because very weak immunoreaction was obtained in tissues from the latter two species. Immunoblots of soluble proteins separated by two-dimensional polyacrylamide gel electrophoresis showed that the sera recognized a family of polypeptides in the adrenal which differed in size, but had almost identical isoelectric points. The patterns of immunoreactive proteins in extracts from the adrenal and pituitary were similar. Only two bands corresponding to the major high molecular weight bands in adrenal could be detected in the hippocampus which appeared to have a lower concentration of antigen. Other brain areas also showed two major immunoreactive proteins, one with a molecular weight similar to chromogranin A, and one smaller. Adrenal chromaffin cells, peripheral noradrenergic nerve axons and terminals in the pineal gland, a proportion of the anterior pituitary cells and the neurosecretory terminals of the posterior pituitary were strongly immunoreactive. In addition, CHR-immunoreactivity was widely distributed in the brain and spinal cord. The reactivity was readily visible in some nerve cell bodies and in well-defined pathways and terminal fibre networks. There were neurons whose perikarya were intensely stained but whose terminal projections appeared to be negative, while in other cases, the terminals appeared rich in CHR, while the perikarya were barely stained. All chromogranin immunoreactivity was abolished by absorption of the sera with a lysate from the chromaffin granules, but was not affected by absorption with Met- or Leu-enkephalin, dynorphin1-17, Met-enkephalin-Arg6-Phe7 or BAM-22P. Electron microscopic experiments revealed that the CHR-reaction in cell bodies was almost exclusively confined to the Golgi apparatus, while in synaptic boutons it was found in large dense-cored vesicles common to many types of terminals. In the hippocampal mossy fibre terminals, the immunoreactive granulated vesicles sometimes appeared to have fused with the plasma membrane of the boutons suggesting that the CHR was being secreted by exocytosis. The CHR-immunoreactivity was found to overlap partially with the distribution of many other neuroactive substances.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Medulla↗
PubMed1984

Biochemical and functional properties of large and small dense-core vesicles in sympathetic nerves of rat and ox vas deferens.

A comparative study of the noradrenaline storing vesicles in vas deferens from ox and rat was performed. Microsomal fractions were subjected to density gradient centrifugation. In rat, noradrenaline and dopamine beta-hydroxylase were mainly present in the upper fractions of the gradient, which is consistent with the predominance of light (small dense-core) vesicles in this species. In ox, noradrenaline, dopamine and dopamine beta-hydroxylase were found in the gradient in a bimodal distribution. This is consistent with the presence of about equal numbers of small and large dense-core vesicles in this species. On the other hand, chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity were only present in the dense (large dense-core) vesicle population. In order to study the capability of light and dense vesicles to synthesize noradrenaline we "pulse-labelled" ox vasa deferentia with [3H]tyrosine. Already 3.5 min after the pulse both types of vesicles contained [3H]noradrenaline and [3H]dopamine. During longer "chase" periods the amount of [3H]dopamine gradually declined. We conclude that dense (large dense-core) vesicles contain chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity whereas light (small dense-core) vesicles are devoid of these components. Both types of vesicles contain dopamine beta-hydroxylase and can synthesize noradrenaline from dopamine under in vivo conditions.

Animals↗
PubMed1984

Exocytotic exposure and retrieval of membrane antigens of chromaffin granules: quantitative evaluation of immunofluorescence on the surface of chromaffin cells.

The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine beta-hydroxylase. With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Ba2+) exhibited much stronger reactions. In all cases the staining appeared as dots and patches. To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescence-activated cell sorter. After stimulation, the average fluorescence intensity of the cell population was enhanced. This increase correlated with the degree of catecholamine secretion. The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues. When stimulated cells were incubated at 37 degrees C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed. Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process. This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine. The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues.

Adrenal Medulla↗
PubMed1984

Isolation and immunological characterization of a glycoprotein from adrenal chromaffin granules.

A glycoprotein (s-GP III) was isolated from the soluble lysate of chromaffin granules by chromatography with immunoaffinity and lectin columns. An identical protein (m-GP III) was shown to be present in the granule membranes. The apparent molecular weight of these glycoproteins as determined by the electrophoresis system of Laemmli (1970) was 43,000 under reducing conditions. In the absence of mercaptoethanol they aggregated to dimers. Antisera were raised against both the soluble and the membrane-bound forms of this glycoprotein. With these antisera GP III was further characterized: Immunoreplicas were obtained after two-dimensional electrophoresis of soluble and membrane-bound proteins of chromaffin granules. GP III was identified as a protein with a rather broad pI (4.6-5.3), indicating microheterogeneity. As shown by subcellular fractionation, m-GP III is specifically confined to chromaffin granules. GP III can therefore be used as a marker for the membranes of these organelles. The soluble form is secreted from adrenal medulla during stimulation with carbamylcholine chloride. An immunologically identical antigen was detected in adeno- and neurohypophysis. The physiological function of GP III is still unknown. It does not demonstrate any of the enzymatic activities so far known to occur in chromaffin granules.

Adrenal Medulla↗
PubMed1983

Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla.

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.

Adenosine Triphosphate↗