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H Winkler

Publications and source records attributed to H Winkler.

At least 109 records · Page 6Linked to original sources
PubMed1995

Biosynthesis of large dense-core vesicles in PC12 cells: effects of depolarization and second messengers on the mRNA levels of their constituents.

mRNA levels of various constituents of large dense-core vesicles were determined in PC12 cells during depolarization and/or in the presence of BayK 8644, forskolin or phorbolester. For the soluble (secretory) proteins of the vesicles the mRNAs of chromogranin A and B, secretogranin II, neuropeptide Y and VGF were analyzed. Depolarization in the presence of BayK induced a strong up-regulation of the messages for chromogranin B, neuropeptide Y and VGF. Addition of forskolin enhanced this response for neuropeptide Y and VGF, phorbolester did the same only for VGF. Partly membrane-bound and membrane-spanning components analyzed were carboxypeptidase H, dopamine beta-hydroxylase and glycoprotein III (clusterin), peptidylglycine alpha-amidating mono-oxygenase and cytochrome b-561, respectively. Changes of mRNAs for these components were in general smaller and delayed. Six days of depolarization caused an up-regulation of glycoprotein III, peptidylglycine alpha-amidating mono-oxygenase and carboxypeptidase H mRNA levels which were not further increased by cyclic AMP and phorbolester. The dopamine beta-hydroxylase message increased after 6 days of depolarization, however, addition of phorbolester reduced this effect. For cytochrome b-561 there was no change after any of the conditions employed. These in vitro results are compared with those obtained for the biosynthesis regulation of large dense-core vesicles under in vivo conditions. It is suggested that in vivo acetylcholine and vasoactive intestinal polypeptide released from splanchnic nerve induce a differential change in the biosynthesis of large dense-core vesicles by acting via calcium and protein kinase A and C.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Effects of haloperidol, clozapine and citalopram on messenger RNA levels of chromogranins A and B and secretogranin II in various regions of rat brain.

We have measured the messenger RNA levels of chromogranins A and B and secretogranin II in various brain regions of rats subchronically treated with various antipsychotic drugs. Since, as shown previously, the messenger RNA levels of these peptides are increased when neurons are stimulated, we hoped to identify by this approach those nuclei which are subchronically influenced by these drugs. The drugs chosen were the neuroleptic halperidol, a blocker of dopamine receptors, the atypical antipsychotic clozapine, which in addition to blocking dopamine receptors also blocks those for serotonin, and citalopram, a specific serotonin reuptake inhibitor. In agreement with previous data on neuropeptide messenger RNAs, we found in the dorsolateral striatum an increase of the secretogranin II messenger RNA levels after haloperidol and a much smaller one after clozapine. In the nucleus accumbens and in the bed nucleus of the stria terminalis, both compounds had a comparable positive effect. These differential effects can be attributed to a different action of these drugs on dopamine receptor subtypes. In the zona incerta, clozapine decreased the secretogranin II and chromogranin A message, whereas in the dorsal raphe it led to an increase. On the other hand, citalopram induced exactly the opposite effects in these two brain regions. This phenomenon can be explained by the differential interaction of these drugs with serotonergic mechanisms. Additional, relatively small changes of the mRNAs were seen in several other brain regions. These results establish that changes in the mRNA levels of the chromogranins are good indicators for the effect of drugs on certain brain nuclei. The concomitant action of haloperidol and clozapine on the limbic regions, i.e. the nucleus accumbens and the bed nucleus of the stria terminalis, points to these brain regions for the antipsychotic action of these two neuroleptics.

Animals↗
PubMed1995

Sustained dopamine release induced by secretoneurin in the striatum of the rat: a microdialysis study.

Secretoneurin (SN) is a neuropeptide derived from secretogranin II that is found in brain and endocrine tissues. The aim of the present study was to determine the influence of this novel peptide on dopamine (DA) release from rat striatum using the microdialysis technique. Rat SN (1-30 mumol/L added to the dialysis buffer) enhanced DA outflow of awake rats in a concentration-dependent way without marked effects on the outflow of 3,4-dihydroxyphenylacetic acid or homovanillic acid. The increase in extracellular DA content caused by the peptide was observed throughout the entire period of administration (up to 4 h). Human SN and its 15-amino-acid C-terminal sequence also increased DA outflow, but the effects were smaller than those of rat SN. Two other peptides derived from secretogranin II were without effect on DA efflux. These results establish that SN has a pronounced effect on DA release under in vivo conditions.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1995

Role of endothelial cells in transplantation.

Endothelial cell activation with accompanying vascular inflammatory changes is considered central to the experimental manifestations of both hyperacute and delayed xenograft rejection responses. Natural xenoreactive antibodies directed at alpha-galactosyl residues of xenogeneic glycoproteins and glycolipids, with associated complement activation via the classical pathway, are considered major immediate mediators of graft endothelial cell injury in the clinically relevant discordant swine to primate combinations. In delayed xenograft rejection processes, where recipients are treated prophylactically to ameliorate these initial events, activation of infiltrating mononuclear phagocytes and natural killer cells are associated with ongoing endothelial cell activation processes, procoagulant generation and vascular thrombosis. Allograft hyperacute rejection is observed when vascularised organs are transplanted to sensitized individuals with high levels of cytotoxic antibodies. Less dramatic forms of humoral allograft rejection (termed accelerated or vascular rejection) and the more common cell-mediated endothelialitis are associated with significant graft damage. Endothelial cell activation is also linked with graft preservation injury, forms of chronic rejection and delayed graft loss. Experimental work is currently being directed at the control of hyperacute rejection, the close understanding of endothelial cell thromboregulation in both transplanted xeno- and allografts and the development of novel therapeutic agents including gene therapy and the possible use of organs from transgenic animals.

Cell Transplantation↗
PubMed1995

[Necrosis of the rectus abdominis muscle. Complication after ilioinguinal approach].

After a car accident, a 19-year-old man had to undergo laparotomy in a local hospital due to intra-abdominal bleeding. A right-sided transrectal incision was chosen, and to improve exposure the rectus muscle was cut horizontally at the cranial end of the incision. Ten days after the accident the patient was transferred to our hospital for stabilization of the pelvic and acetabular fracture on the right side. An ilioinguinal approach was chosen. Two days later the abdominal cavity was opened due to necrosis of the cranial part of the rectus abdominis muscle. Following a 10-day period without pathological symptoms, suddenly sepsis developed. Ultrasound showed a lot of fluid in the rectus sheath; no muscle structure could be identified. The operation showed total necrosis of the rectus muscle due to thrombotic occlusion of the inferior epigastric artery. The standardized tourniquets around the vessels used during the ilioinguinal approach seemed to be responsible for the rectus muscle necrosis. Therefore, if a combined intraabdominal and pelvic approach has to be used, median laparotomy is the better choice, because no risk is posed to the blood supply of the rectus muscle, in contrast to the ilioinguinal approach. The problem is discussed in detail.

Abdominal Injuries↗
PubMed1994

Human and rat primary C-fibre afferents store and release secretoneurin, a novel neuropeptide.

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre-treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C-) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin-immunoreactivity (IR) in the spinal cord and, lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post-mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin-IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (< 25 microns) SgII-mRNA-containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin-IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin-treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin-IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin-sensitive, primary afferent (C-fibre) neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Bovine chromaffin cells: studies on the biosynthesis of phospholipids in chromaffin granules.

We have studied the biosynthesis of chromaffin granules by labelling primary cultures of bovine chromaffin cells with either [35S]methionine or various precursors for lipids. After labelling the cells were subjected to subcellular fractionation including density gradient centrifugation. After [35S]methionine significant label (mainly represented by labelled chromogranin A) was found in the soluble proteins of chromaffin granules, whereas the membranes were relatively little labelled. However incorporation into membrane bound dopamine beta-hydroxylase and cytochrome b-561 could be demonstrated. Neither of the used lipid precursors ([3H]glycerol, [3H]choline, [3H]palmitic acid or [3H]arachidonic acid) was incorporated to any significant extent into the soluble components of chromaffin granules. Thus there is no evidence that this secretory compartment contains any lipids or acylated proteins. Incorporation of lipid precursors into the membranes of chromaffin granules was apparently low. After short chases labelled lysolecithin was not present in these organelles. However with prolonged chase times labelled lysolecithin, apparently appeared in chromaffin granules irrespective of whether the cells were stimulated or not. We can conclude that the reusable membranes of chromaffin granules have a very low lipid turnover. Lysolecithin is not transferred into these organelles during biosynthesis but is formed in them during their long life span. This formation of lysolecithin is independent of stimulation of these cells and therefore unlikely to be involved in exocytosis.

Adrenal Medulla↗
PubMed1994

Mössbauer and magnetic susceptibility studies on iron(II) metallothionein from rabbit liver. Evidence for the existence of an unusual type of [M3(CysS)9]3- cluster.

The magnetic properties of the Fe(II)-binding sites in Fe(II)7-metallothionein (MT) have been studied using Mössbauer spectroscopy and magnetic-susceptibility measurements. In agreement our previous results, simulation of the Mössbauer spectra showed the presence of paramagnetic and diamagnetic subspectra in the ratio 3:4. By comparison with Mössbauer spectra of the inorganic adamantane-like (Et4N)2[Fe4(SEt)10] model compound, the diamagnetic component in Fe(II)7-MT has been assigned to a four-metal cluster in which there is antiferromagnetic coupling between the high-spin Fe(II) ions. It is suggested that the organization of this cluster is similar to that determined in the three-dimensional structure of the protein, containing diamagnetic Zn(II) and/or Cd(II) ions. From magnetic-susceptibility studies, an average magnetic moment of approximately 8.5 microB was obtained for the three remaining bound Fe(II) ions, responsible for the paramagnetic component observed in the Mössbauer studies. This value is slightly lower than that for three completely uncoupled Fe(II) ions, suggesting the existence of a three-metal cluster within which there is weak exchange coupling between adjacent Fe(II) ions. The spin-Hamiltonian formalism including, besides zero-field and Zeeman interaction, also exchange interaction among the three Fe(II) ions in the three-metal cluster, H = -J12 (S1.S2)-J23 (S2.S3)-J13 (S1.S3), was applied to simulate both magnetic-Mössbauer and magnetic-susceptibility data. Reasonable fits were achieved only with values magnitude of J12 = magnitude of J23 = magnitude of J13 = magnitude of J < 1 cm-1. Such a situation could not be reconciled with the chair-like geometry of the [M3(CysS)9]3- cluster determined with paramagnetic metal ions, where significantly stronger coupling would be anticipated (magnitude of J = 50-70 cm-1). However, modest exchange-coupling properties have been reported for a number of crystallographically characterized trinuclear [Fe3(SR)3X6]3- clusters (X = Cl, Br; R = Phe, p-tolyl, 2,6-Me2C6H3) distinguished by the preferential formation of a planar Fe3(mu 2-SR)3 ring [Whitener, M. A., Bashkin, J. A., Hagen, K. S., Girerd, J.-J., Gamp, E. Edelstein, N. & Holm, R. H. (1986) J. Amer. Chem. Soc. 108, 5607-5620]. It is therefore more likely that a pseudo-planar geometry rather than a chair-like geometry is present in the Fe3 cluster of Fe(II)7-MT. This would represent the first example of structural differences on binding divalent metal ions to this protein.

Adamantane↗
PubMed1994

Distribution of secretoneurin immunoreactivity in the spinal cord and lower brainstem in comparison with that of substance P and calcitonin gene-related peptide.

Secretoneurin is a peptide of 33 amino acids generated in brain by proteolytic processing of secretogranin II. The distribution of this newly characterized peptide was investigated by means of immunocytochemistry and in situ hybridization in the spinal cord and lower brainstem of the rat. The staining pattern of secretoneurin immunoreactivity (IR) was compared to that of substance P (SP) and calcitonin gene-related peptide (CGRP) in adjacent sections. A high density of secretoneurin-IR fibers and terminals was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord at all levels, around the central canal, and in the sympathetic and parasympathetic areas of the lateral cell columns. The ventral horn displayed a low to moderate density of secretoneurin-IR. The highest number of secretogranin II mRNA-containing cells was found in lamina II of the dorsal horn and in neurons of the dorsal root ganglia. In the white matter, secretoneurin-IR was most prominent in the dorsolateral part of the lateral funiculus and in the tract of Lissauer. The distributions of secretoneurin-IR and SP-IR were strikingly similar. CGRP-IR and secretoneurin-IR overlapped in the outer laminae of the dorsal horn, in the lateral cell column, and probably in some motoneurons. This study establishes that, like SP and CGRP, secretoneurin is a peptide highly concentrated in the terminal field of primary afferents and in sympathetic and parasympathetic areas. Thus secretoneurin might be involved in the modulation of afferent transmission.

Animals↗
PubMed1994

Selective inhibition of E-selectin, ICAM-1, and VCAM in endothelial cells.

Endothelial cells, as they normally exist in the vasculature as quiescent cells, perform several functions. In an inflammatory response, endothelial cells are activated to up-regulate a number of genes, including E-selectin (ELAM-1), VCAM-1, ICAM-1, interleukin (IL)-1, IL-8 and plasminogen activator inhibitor-1 (PAI-1). Very little is known about factors that regulate the activation process. We describe here that a heat-stable protein, normally present in the alpha-globulin fraction of serum, inhibits induced expression of E-selectin, ICAM-1, and VCAM-1 in vitro and also impedes the accumulation of mRNA for these molecules. Inhibition of E-selectin, the only gene tested in this respect, is at the level of transcription. At the same time, the alpha-globulins do not, under the same conditions, repress mRNA accumulation for IL-1, IL-8, or PAI-1. The effect of the inhibitor does not relate to constraints on function of nuclear-factor kappa B, the induced activity of which is not interfered with at the early time points at which the suppression of these three genes is seen.

Alpha-Globulins↗
PubMed1994

Release of secretoneurin and noradrenaline from hypothalamic slices and its differential inhibition by calcium channel blockers.

Secretoneurin is a newly discovered peptide found in high concentrations in brain. We have studied the release of secretoneurin and noradrenaline from superfused hypothalamic slices from rat brain. Both electrical stimulation and potassium induced depolarisation released secretoneurin and noradrenaline from these slices in a calcium-dependent manner. Electrical stimulation caused a preferential release of noradrenaline when compared to the secretion elicited by high potassium. The time course of secretoneurin release was more protracted than that of noradrenaline. The calcium channel blocker omega-conotoxin inhibited only the electrically induced release of noradrenaline, whereas nifedipine inhibited only that of secretoneurin. These results establish that secretoneurin is secreted from neurons. Inhibition of this release by nifedipine is consistent with the concept that secretion from large dense core vesicles occurs at sites different from that of small vesicles and depends on calcium influx via L-type calcium channels.

Animals↗
PubMed1994

Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency.

The biochemical properties of erythrocyte pyruvate kinase (PK) together with mutations found in the coding sequence of the R-PK gene in five patients with severe hemolytic anemia due to PK deficiency are described. The enzyme variants were designated PK 'Mosul' (homozygote), PK 'Bukarest', PK 'Hamburg', PK 'Köln', and PK 'Essen' (compound heterozygote). PK 'Mosul' showed normal positive cooperative substrate binding, PK 'Bukarest' exhibited non-cooperative behavior, and PK 'Hamburg' and PK 'Köln' displayed mixed cooperativity, whereas PK 'Essen' was negative cooperative. PK 'Mosul' was found to be homozygous for the mutation 1151 ACG to ATG, resulting in an amino acid substitution 384 Thr to Met. In one allele of PK 'Bukarest' a single nucleotide substitution GAG-TAG was found at nucleotide 721, causing a change of 241 Glu to a chain termination codon (PK 'Bukarest'). Additionally, in the second allele of this patient a point mutation at position 1594 (CGG-TGG) occurs, changing 532 Arg to Trp (PK 'Bukarest'). Direct sequencing showed the heterozygosity of the patient's mother (PK 'Bukarest'/normal) at position 721 and of the patient's father (PK 'Bukarest'/normal) at position 1594. A point mutation at position 1529 (CGA-CAA), causing an amino acid substitution 510 Arg-Gln, was identified in PK 'Hamburg' and PK 'Köln'. The second mutation in these variants was not detected. In PK 'Essen' no mutation in the coding sequence was found at all. Screening for the mutation at position 1529 in further compound heterozygote patients and in normal subjects of Western European origin showed that this exchange is a common mutation responsible for PK deficiency in this population.

Adult↗
PubMed1994

[Surgical treatment of diaphyseal tibial fractures. Choice of procedure and results of treatment of 187 fractures].

From 1988 to 1990 a total of 187 fractures of the tibia (92 compound, 95 closed fractures) were treated with a primary osteosynthesis. In 102 fractures an external fixation was performed, 85 internal fixations were divided into 58 intramedullary nails and 27 plates. In a follow-up study the fracture healing was analysed, 90% of the patients were examined about 18 months after the accident. The mean healing time was between 12 weeks (internal fixation) and 16 weeks (external fixateur). After primary Fixateur externe 54 were treated by secondary internal fixation, intramedullary nailing was the method of choice. Contamination rate of the tibia, taken by an intraoperative wound swab before nailing was 30%. Infection occurred in 3.2%, non union and refractures in 1.5% and 1%. Over all 80% excellent and good results were found after consolidation. 10% fair and 10% bad results including three amputations after III degrees compound fractures and three death after polytrauma.

Adolescent↗
PubMed1994

Experience with the pinless fixator in the treatment of fractures of the lower leg.

This is a report of the experience we have gathered in the application of the pinless fixator in cases of tibial fracture with soft tissue damage. In the Berufsgenossenschaftlichen Unfallklinik Ludwigshafen (Clinic for Trauma Surgery) 12 diaphyseal fractures of the tibia were stabilized with the pinless fixator in 1991. In all cases, conversion to an intramedullary nail was intended from the very beginning and was performed in 11 cases. In one case, a conventional external fixator had to be selected instead. The injuries included first and second degree open and closed fractures, mostly from group A of the AO classification. At conversion four bacteriological smear tests from the medullary cavity proved positive. In four cases, transitory peroneal deficiencies were diagnosed. When investigating their cause, it seemed that the present geometry of the pinless arms might be responsible for pressure damage to the soft tissues. Possibly this difficulty could be solved by altering the configuration of these arms. The pinless external fixator has definitely proved its usefulness when later conversion to an intramedullary nail is intended since the final fixation can be performed with the pinless device still in place.

Adult↗
PubMed1994

3D reconstruction by combining data from sections cut oblique to different unit cell axes.

Oblique section reconstruction (OSR) can produce a 3D image from a single micrograph of a section through a 2D or 3D crystal. Resolution, however, is limited in a direction perpendicular to the section plane. When data from sections cut at different orientations are combined, the inherent resolution limitation for single sections can be overcome. In the extreme, combination of sections cut oblique to different principal planes of the unit cell can produce a 3D reconstruction with isotropic resolution. The merging of data from different sections requires determination of section thickness and orientation. A method for section thickness determination is described that is generally applicable event to space groups with little or no symmetry. In addition, an approach has been developed to determine rotational orientation within the hexagonal lattice of insect flight muscle. Finally, an iterative least-squares fitting method is applied to determine a 3D set of deconvoluted structure factors from the merged data.

Adenylyl Imidodiphosphate↗
PubMed1994

Molecular characterization of immunoreactivities of peptides derived from chromogranin A (GE-25) and from secretogranin II (secretoneurin) in human and bovine cerebrospinal fluid.

Chromogranin A and secretogranin II are members of the so-called chromogranins, the acidic proteins stored in neuroendocrine large dense-core vesicles. We characterized chromogranin A and secretogranin II immunoreactivities in cerebrospinal fluid by radioimmunoassays using synthetic peptides derived from these components (GE-25 for chromogranin A and secretoneurin for secretogranin II). In lumbar cerebrospinal fluid, high levels (more than 1000 fmol/ml) of these two components were found, whereas in ventricular cerebrospinal fluid the secretoneurin levels were relatively low. The cerebrospinal fluid/serum ratio for secretoneurin was close to 170. High-performance liquid chromatography revealed that in both cerebrospinal fluid and extracts from human brain secretoneurin was the predominant immunoreactive component. In cerebrospinal fluid chromogranin A immunoreactivity was present as intermediate-sized peptides with little intact chromogranin A and free GE-25 peptide. In human brain samples smaller peptides including GE-25 were more predominant. Analogous findings for secretoneurin and chromogranin A were obtained for bovine brain samples. We can conclude that chromogranins are present in cerebrospinal fluid in concentrations much higher than those of classical neuropeptides also stored in large dense-core vesicles. Therefore, their degree of proteolytic processing can be analysed with small samples of cerebrospinal fluid. A possible disturbance of proteolytic processing in large dense-core vesicles in various pathological conditions can now be discovered.

Adult↗
PubMed1994

Large dense-core vesicles in rat adrenal after reserpine: levels of mRNAs of soluble and membrane-bound constituents in chromaffin and ganglion cells indicate a biosynthesis of vesicles with higher secretory quanta.

Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense-core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense-core vesicles (chromogranins A and B, secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 215-857% after 1-3 days of drug treatment. For partly membrane-bound components (dopamine beta-hydroxylase, prohormone convertase 2, carboxypeptidase H, and peptidylglycine alpha-amidating monooxygenase) the changes ranged from 182 to 315%, whereas for glycoprotein III and for intrinsic membrane proteins (cytochrome b561 and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y, secretogranin II, carboxypeptidase H, and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Long-lasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides.

Adrenal Glands↗