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B Zimmermann

Publications and source records attributed to B Zimmermann.

At least 19 recordsLinked to original sources
PubMed1999

Purification procedure and monoclonal antibodies: two instruments for research on vertebrate porins.

On Western blots of skeletal muscle preparations of different vertebrate classes, four monoclonal anti-human type 1 porin antibodies recognize one single band of either 30.5 or 31 kDa, respectively. To confirm that it is eukaryotic porin which is labeled by the antibodies, we used a purification procedure developed for human type 1 porin for porins from skeletal muscle of shark, frog, and turkey. Applied to different mammalian species and tissues, this procedure exclusively provides type 1 porin. However, applied to shark skeletal muscle, it provides two porin isotypes in nearly equal amounts. In the case of frog skeletal muscle, the procedure provides mainly type 2 porin and a lower amount of type 1 porin. Applied to turkey skeletal muscle, the method provides exclusively type 2 porin. As demonstrated by two-dimensional Western blots, both shark and frog porin isotypes and the turkey type 2 porin are recognized by our antibodies. Furthermore, we elucidated the entire amino acid sequence of frog type 2 porin.

Amino Acid Sequence↗
PubMed1999

The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland.

Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel.

Animals↗
PubMed1999

PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I.

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.

Amino Acid Sequence↗
PubMed1999

Impaired neutrophil exocytosis in patients with severe pneumonia.

OBJECTIVE: Polymorphonuclear neutrophils (PMN) are one of the major effector cells of pulmonary defence against bacterial infection. To determine whether neutrophil function is impaired in patients with severe pneumonia, we assessed the two main partial functions exocytosis and oxidative response (ROS production) in isolated neutrophils from the peripheral venous blood of pneumonia patients and healthy volunteers. In addition, pulmonary neutrophils and peripheral neutrophils were compared in pneumonia patients. PATIENTS AND METHODS: Twenty-one patients with severe pneumonia were enrolled in the study. Eleven patients were mechanically ventilated, ten patients breathed spontaneously. For comparison, ten healthy adults were studied. The release of two markers of neutrophil exocytosis, lactoferrin and myeloperoxidase (MPO), with and without stimulation by phorbol-myristate-acetate (PMA), was determined using immunoluminometric assays. ROS production was quantified using luminol-enhanced chemiluminescence. In addition, the clinical severity of pneumonia was correlated to neutrophil exocytosis. RESULTS: With regard to blood neutrophils, both basal and PMA-stimulated exocytosis were significantly impaired in pneumonia patients compared to healthy volunteers (basal lactoferrin secretion in pneumonia patients: 0.25+/-0.36 pg/PMN versus controls: 1.17+/-0.78 pg/PMN, p<0.01). In contrast, both basal and PMA-stimulated ROS production were increased in patients compared to controls (spontaneous chemiluminescence in pneumonia patients: 13.6x10(5) cpm versus controls: 5.5x10(5) cpm). In pneumonia patients, the pulmonary neutrophils released significantly more lactoferrin, MPO and ROS compared to blood neutrophils (basal lactoferrin secretion of pulmonary neutrophils: 1.19+/-1.55 pg/PMN; p<0,01). However, after stimulation with PMA the exocytosis of pulmonary and blood neutrophils was similar. The severity of pneumonia and prognostic indices like albumin were inversely correlated to the release of lactoferrin in blood neutrophils (p<0,05). CONCLUSION: In patients with severe pneumonia, the exocytosis of blood neutrophils was significantly impaired. In contrast to this, the oxidative response was increased. Impaired bone marrow maturation of neutrophils during severe infection, perhaps due to shortened maturation time, could explain these findings.

Adult↗
PubMed1998

Calcium store depletion activates two distinct calcium entry pathways in secretory cells of the blowfly salivary gland.

Ca2+ influx into secretory cells of the intact salivary gland of the blowfly Calliphora erythrocephala elicited by the agonist 5-hydroxytryptamine (5-HT) or the Ca2+ uptake inhibitor thapsigargin was studied by using Fura-2 and digital fluorescence imaging and by recordings of the transepithelial potential. Application of saturating [5-HT] in the absence of Ca2+ (Ca2+o) from the bathing saline did not affect the initial Ca2+ transient but greatly attenuated the subsequent sustained Ca2+ elevation observed in the presence of Ca2+o demonstrating that the latter component of the [Ca2+]i response is largely dependent on Ca2+ entry across the baso-lateral plasma membrane. La3+ or Gd3+ (10 microM) mimicked the effects of the withdrawal of Ca2+o. Experimental attempts temporally to uncouple 5-HT stimulation and Ca2+ influx by withdrawal of Ca2+o during agonist application revealed a second Ca2+ entry pathway. This pathway was insensitive to 10 microM La3+ and produced transient [Ca2+]i increases whose amplitudes were a function of the [5-HT] during the preceding stimulation and that were selectively suppressed by 50 microM SK&F 96365. Both (10 microM) La(3+)-insensitive [Ca2+]i transients and (10 microM) La3+ inhabitable tonic [Ca2+]i increases could be sequentially activated in the presence of 5-HT or thapsigargin (1 microM). These results indicate that Ca2+ store depletion by 5-HT or thapsigargin activates two distinct store-operated Ca2+ entry pathways, one of which supports tonic [Ca2+]i increases. The other is transiently activated, even under conditions that prohibit store refilling and does not significantly contribute to the [Ca2+]i responses evoked by saturating 5-HT concentrations.

Animals↗
PubMed1998

Inhibition of PrKX, a novel protein kinase, and the cyclic AMP-dependent protein kinase PKA by the regulatory proteins of adeno-associated virus type 2.

Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40. Expression of these nonstructural proteins affects cell growth and gene expression through processes that have not yet been characterized. Using a yeast two-hybrid screen, we have demonstrated that a stable interaction occurs between the viral proteins Rep78 and Rep52 and the putative protein kinase PrKX, which is encoded on the X chromosome. The stability and specificity of the Rep-PrKX interaction were confirmed by coimmunoprecipitation of complexes assembled in vitro and in vivo. Overexpressed PrKX, which was purified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inhibited by stoichiometric amounts of Rep52 and weakly inhibited with Rep68, which lacks the carboxy-terminal sequence contained in Rep52. Similarly, a stable interaction was observed with Rep78, which also contains the carboxy-terminal sequence of Rep52. A stable interaction and inhibition were also observed between Rep52 and the catalytic subunit of PKA. By using surface plasmon resonance and kinetic studies, Kis of approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX, respectively. Thus, Rep52 but not Rep68 can significantly inhibit the trans- and autophosphorylation activities of these kinases. The biological effects of Rep78-specific inhibition of PKA-responsive genes are illustrated by the reduction of steady-state levels of cyclic AMP-responsive-element-binding protein and cyclin A protein.

Amino Acid Sequence↗
PubMed1997

Serotonin-induced intercellular calcium waves in salivary glands of the blowfly Calliphora erythrocephala.

1. Blowfly salivary glands have been used extensively as a model system for the analysis of inositol phosphate-dependent signal transduction. To detect and characterize changes in intracellular free calcium ([Ca2+]i) that might be expected to be triggered by stimulation with serotonin (5-HT), we have carried out digital calcium-imaging experiments on intact glands using the Ca2+-sensitive dye fura-2. 2. 5-HT (1-10 nM) induced repetitive transient increases in [Ca2+]i, i.e. Ca2+ spikes whose frequency was a function of agonist concentration (EC50 = 2.8 nM). 3. Pre-incubation in EGTA decreased the frequency but did not inhibit spiking. Thapsigargin abolished periodic spike activity indicating that the [Ca2+]i rise results from Ca2+ release. Neither caffeine (10 mM) nor ryanodine (10 and 50 microM) induced increases in [Ca2+]i. 4. Oscillatory activity in individual cells was synchronized by regenerative intercellular Ca2+ waves that propagated over distances greater than 400 microm. Colliding waves annihilated each other. 5. Desynchronization of the oscillation pattern by 100 microM 1-octanol suggests the involvement of gap junctions and an intracellular messenger in wave propagation. 6. Local stimulation of glands elicited [Ca2+]i elevations in the stimulated area, but not in adjacent cells, indicating that local increases in [Ca2+]i are not sufficient to trigger Ca2+ waves. However, local stimulation was capable of evoking propagating Ca2+ waves when combined with low-dose 5-HT stimulation of the whole gland. 7. The data are consistent with the hypothesis that: (1) Ca2+ acts as the intercellular messenger and modulates its own release via positive and negative feedback on the inosital 1,4,5-trisphosphate (InsP3) receptor, and (2) sensitization of the InsP3 receptor to Ca2+ by InsP3 is required for the propagation of intercellular Ca2+ waves, as proposed for intracellular Ca2+ waves in Xenopus oocytes.

Animals↗
PubMed1997

Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface.

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.

Catalysis↗
PubMed1997

Effects of all-trans-retinoyl-beta-D-glucuronide and all-trans-retinoic acid on chondrogenesis and retinoid metabolism in mouse limb bud mesenchymal cells in vitro.

Retinoids, derivatives of vitamin A, are essential for many vertebrate functions. Furthermore, several drugs of this class of compounds are valuable in the treatment of certain forms of skin disorders and cancer. However, the therapeutic application of retinoids is limited by their teratogenic potency. The limbs are important sites of retinoid-induced malformations in rodents. Therefore, organoid cultures of limb bud mesenchymal cells have been established for screening of the teratogenic potency of retinoids. We have now applied this system to compare the effects of all-trans-retinoyl-beta-D-glucuronide (all-trans-RAG) with those of all-trans-retinoic acid (all-trans-RA) on chondrogenesis, as assessed by the Alcian blue binding assay and by electron microscopic evaluation including quantitative morphometric analysis. First data of retinoid toxicokinetics in the culture media as well as retinoid concentrations in the cultured mesenchymal limb bud cells were established. While all-trans-RA inhibited chondrogenesis at 10(-7) M by ca. 50%, tenfold higher concentrations of all-trans-RAG were necessary to obtain the same effect. This difference reflects the ratio of RA isomers which were found in the medium after incubation with either all-trans-RAG or all-trans-RA. A pulse experiment (10(-5) M all-trans-RAG or all-trans-RA for the first 2 h of a 6-day incubation period) demonstrated inhibition of chondrogenesis with all-trans-RA, but not with all-trans-RAG. The data indicate that RAG inhibits chondrogenesis upon hydrolysis to RA. Surprisingly, the rather polar RAG isoforms were extensively accumulated in the limb bud mesenchymal cells when compared to the medium. Both all-trans-RAG and all-trans-RA also induced a large increase of retinyl ester concentrations in the chondrocytes compared to vehicle-treated cells. This finding further supports a recent suggestion that RA regulates retinol metabolism via feedback inhibition of retinol oxidation and stimulation of the esterification of retinol.

Alcian Blue↗
PubMed1997

Endoproteolysis of glucagon-like peptide (GLP)-1 (7-36) amide by ectopeptidases in RINm5F cells.

This study concerns whether the pancreatic beta cell expresses cell-surface ectopeptidases that are capable of proteolysis of peptide hormones and neuropeptides that modify glucose-dependent insulin release. These biochemical investigations of the RINm5F cell line found that these cells express ectopeptidases. We have characterized the limited endoproteolysis of GLP-1 (7-36) amide that occurs in the presence of RINm5F plasma membranes. The products and the sensitivity to specific peptidase inhibitors of the proteolysis is characteristic of neutral endopeptidase (NEP) 24.11. Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), amylin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), and exendin-4 also undergo proteolysis in the presence of RIN cell membranes. NEP 24.11-activity in RIN cell membranes was confirmed using a specific fluorogenic assay, by histochemistry, and by comparison with the recombinant enzyme with respect to the kinetics of proteolysis of GLP-1 (7-36) amide and of a fluorogenic substrate. Specific fluorogenic assays revealed the presence of aminopeptidase N and the absence of aminopeptidase A and of dipeptidylpeptidase IV.

Amino Acid Sequence↗
PubMed1997

[Colloid osmotic pressure and neonatal respiratory distress syndrome].

BACKGROUND: The interest of studying colloid osmotic pressure (COP) in neonates is based on a possible relation between low COP and severity of neonatal respiratory distress. POPULATION AND METHODS: Because this relation has not been accurately established, COP was prospectively measured at birth in 84 infants: 35 with respiratory distress syndrome treated with mechanical ventilation (group RD+: GA = 34.5 +/- 3.5 weeks; BW = 2,190 +/- 830 g); 49 free of severe respiratory disease (group RD-: GA = 37.5 +/- 2.5 weeks; BW = 2,720 +/- 655 g). RESULTS: On day one after birth COP was significantly lower in the RD+ group (15.0 +/- 2.2 mmHg) as compared with that in the RD- group (17.9 +/- 2.5 mmHg), and was negatively correlated with mean oxygenation index (OI) on day one, with the highest OI over the first five days of life, and with total duration of oxygen support (P < 0.05). COP was significantly reduced in the neonates with systemic hypotension on day one (P < 0.05). CONCLUSIONS: These results confirm a correlation between COP at birth and incidence and severity of neonatal respiratory distress. The relationship between low COP and systemic hypotension suggests an important role of COP in the ability of maintaining volemia. This study stresses the strong relationship between water homeostasis and neonatal respiratory distress. Nevertheless, it does not allow to conclude that modifying COP is necessary to improve neonatal respiratory disease.

Birth Weight↗
PubMed1997

[Treatment of neonatal convulsions].

Three important measures are concommitantly recommended in the treatment of neonatal seizures. 1) The maintenance of vital functions by supplying oxygen and using mask ventilation in addition to cardiac monitoring and placing an intravenous line. 2) The search for an etiology through metabolic and infectious laboratory tests. In many cases this will lead to an etiological treatment. 3) The specific treatment of the seizures: intravenous or intrarectal diazepam is the first line treatment. Phenobarbital, then phenytoin and clonazepam are used when diazepam is unsuccessful. Indications and duration of a secondary prophylactic treatment are controversial and must be discussed on the basis of the electroencephalographic data and the risk factors for epileptic seizures during the neonatal period and at 3 months of age.

Anticonvulsants↗
PubMed1997

The rewards outweigh the efforts: breastfeeding outcomes for mothers of preterm infants.

This study describes the rewards and efforts of breastfeeding for mothers of preterm infants. Using a semi-structured interview guide, 20 mothers of preterm infants were interviewed in their homes approximately 1 month after infant discharge from a Level III NICU. Mothers described the following rewards of breastfeeding: knowing they were providing the healthiest nutrition for the infant, enhancing closeness between the mother and infant, perceiving infant contentment and tranquility during breastfeeding, providing convenience for the mother, and giving the mother a tangible claim on the infant. Most mothers identified some "efforts" associated with breastfeeding their preterm infants, but indicated that overall, breastfeeding was a rewarding experience. These data provide scientific support for the promotion and facilitation of breastfeeding for mothers of preterm infants, in that mothers perceive specific emotional advantages that they relate to the breastfeeding experience.

Adult↗
PubMed1997

Substance P induces the secretion of gelatinase A from human synovial fibroblasts.

We investigated the secretion of the matrix metalloproteinases, interstitial collagenase (matrix metalloproteinase-1), gelatinase A (matrix metalloproteinase-2) and stromelysin-1 (matrix metalloproteinase-3) in human synovial fibroblasts after stimulation with the neuropeptide substance P. Human synovial fibroblasts were stimulated with substance P or interleukin-1 beta (IL-1 beta). In the cell culture media gelatinase A, interstitial collagenase and stromelysin-1 were identified and their activities towards different substrates were determined. Substance P in synovial fibroblasts induced an increase in the overall matrix metalloproteinase activity towards the dinitrophenyl-labelled peptide by 85%, against an increase of 124% after stimulation with IL-1 beta. In case of substance P stimulation, the increase in activity reflects a significantly enhanced secretion of gelatinase A, whereas no significant increase of stromelysin-1 and collagenase secretion could be observed. The matrix metalloproteinase pattern showing the highest gelatinase A secretion was obtained after stimulation with substance P. This pattern was very pronounced and differed very clearly from the pattern seen after IL-1 beta stimulation which caused a significant rise in collagenase and stromelysin-1 activity. We assume that distinct stimulation pathways are involved and that the neuropeptide (substance P), which is always present in the inflamed joint, plays its own and separate role in proliferative processes leading to the cartilage destruction.

Blotting, Western↗
PubMed1997

Relapse of polymyositis after prolonged remission.

We describe the case of a patient with polymyositis and anti-Jo-1 antibodies who experienced a relapse of his inflammatory muscle disease after 7 years of clinical remission. Our review of the literature shows that remission is achieved in 25-68% of patients treated with high dose corticosteroids as part of their initial therapy. Relapse rates after complete remission vary from 6 to 43% in the few studies where this information is available. Late relapses after initial remission appear to be unusual, but may be more frequent in patients like this one with antisynthetase syndrome.

Antibodies, Antinuclear↗