Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

The Tol-Pal system of Acinetobacter baumannii is important for cell morphology,antibiotic resistance and virulence

International Microbiology - Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions. This Gram-negative bacterium is one of the most...     

Translocation of precytochrome C2 into intracytoplasmic membrane vesicles of Rhodobacter capsulatus requires a peripheral membrane protein

Rhodobacter capsulatus is a member of the group α-purple bacteria which are closely related to the ancestral endosymbiont that gave rise to mitochondria. It has therefore been hypothesized that the molecular mechanisms governing protein export in α-purple bacteria have been conserved during the evolution of mitochondria. To enable analysis of protein export in α-purple bacteria we describe here the development of a homologous cell-free synthesis/export system consisting entirely of components of R. capsulatus. Translocation of precytochrome C₂ into intracytoplasmic membrane vesicles of this organism was found to require the proton-motive force and proceed at a significantly higher efficiency when membranes were present during protein synthesis. Furthermore, we show that, in this cell-free system, translocation depends on a preparation of peripheral membrane proteins Which do not possess detectable SecA- and SecB-like actvities.     

Solid-phase synthesis of triple-helical collagen-model peptides

Summary The triple-helical conformation of collagen has been proposed to be important for mediation of cellular activities, such as adhesion and activation, extracellular matrix assembly, and enzyme function. We have developed synthetic protocols that allow for the study of biological activities of specific collagen sequences in triple-helical conformation. These methods primarily involve solid-phase assembly and covalent linkage of three peptide chains. The resultant triple-helical peptides have sufficient thermal stabilities to permit structural and biological characterization under physiological conditions. The present article critically reviews the various approaches for constructing synthetic triple-helices.This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes

Recent immunocytochemical studies have shown that placental villous trophoblasts contain the high molecular weight cytokeratin (CK) proteins 5/6 and 17. In the case of CK 17, trophoblastic immunostaining was positive in villi covered by fibrinoid. CKs 5/6 and 17 are expressed by hyperproliferative cells. The aim of this investigation was to examine the location of these CKs in placental infarcts, known to be demarcated by fibrinoid and hyperproliferative trophoblasts. The results were compared with those obtained by immunostaining against Ki-67, tenascin and α1-, α6- and β1- integrins, which are involved in cell proliferation, differentiation and regenerative processes. Furthermore, the expression of the single CKs 7, 8, 10, 13, 14, 18 and 19 was investigated by immunocytochemistry and immunoblotting. While low and high molecular weight CKs were present in villous and extravillous trophoblasts, only low molecular weight CKs were detected in vascular and extravascular placental smooth muscle cells. Placental infarcts revealed different immunoreactivities in the infarct margin and centre: high molecular CKs, tenascin, Ki-67 and oncofoetal fibronectin predominated in the infarct margin, low molecular CKs, fibrin and integrins in the centre. The expression of tenascin and a defined change in the expression of CK 17 indicates villous repair and hyperproliferative mechanisms in placental infarcts. This revised version was published online in November 2006 with corrections to the Cover Date.     

Epidermal fine structure of the toe tips of Sphaerodactylus cinereus (Reptilia, Gekkonidae)

This paper deals with epidermal structures of the sphaerodactyline gecko Sphaerodactylus cinereus : adhesive pads, cutaneous sensilla and intraepidermal axon terminals.
The adhesive pad is restricted to a single terminal scale and bears approximately 6,000 setae. The setae are complex, hair-like structures which branch and sub-branch up to five times. The terminal ends are shaped like inverted cones. They provide the friction which enables the gecko to walk even on vertical glass-plates.
Cutaneous sensilla of supposed mechanoreceptive function are found in groups of three or four at the anterior free edge of all dorsal and distal scales of the digit. The sensillum consists of a circular platelet in an epidermal depression bordered by an annular furrow and two or three bristles in a central position.
Discoid axon terminals in the digital scales are located between relatively stiff structures: the corneous layers of the epidermis and a layer of tonofibrillar bundles. The axon terminals are hypothesized to be sensitive to the internal pressure depending on hyperextension of the toe.     

Paddle cilia and discocilia — Genuine structures?

Kinocilia of epidermal sensory cells in fixed marine Turbellaria often terminate as flattened biconcave discs. The distal part of the ciliary axoneme curves back upon itself forming a 360 degree loop which is enveloped by the plasmalemma. In living animals this structure can be induced by the addition of sodium cacodylate, monobasic sodium phosphate, dibasic sodium phosphate, sucrose, calcium chloride, or formaldehyde to the sea water. Specimens treated with sodium chloride, glutaraldehyde, or osmium tetroxide do not show modified cilia. In animals prepared for EM at low temperature and with a buffered hypotonic fixative less kinocilia are modified than in animals treated with a buffered iso- or hypertonic fixative and at a higher temperature. It is assumed that the unusually shaped cilia, described as "paddle cilia" or "discocilia" in other invertebrates, do not represent a genuine but an artificial structure.     

Postembedding immunohistochemical demonstration of antigen in experimental polyarthritis using plastic embedded whole joints

Summary A method is presented for the immunohistochemical demonstration of antigens in whole undecalcified joints of small laboratory animals. With this method of tissue preparation, involving embedding in a medium mainly based on 2-hydroxyethyl methacrylate, preservation of antigenicity is satisfactory. Antigens can be demonstrated in 2 m sections by either immunofluorescence or immunoperoxidase and an indirect technique. Therefore in addition to the morphological analysis of joint alterations in experimental polyarthritis, there is now an opportunity to trace the inciting antigen and to study in parallel the enzymatic equipment of the cells involved, using consecutive sections from a single block of tissue.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 54, C2     

Monociliary receptors in interstitial Proseriata and Neorhabdocoela (Turbellaria Neoophora)

Summary The ultrastructure of monociliary receptors in 10 species of the Proseriata and Neorhabdocoela is described, with particular reference to the epidermal dendritic part.Sensory cells with a single kinocilium situated at the level of the distal epidermis membrane are considered as mechano- or chemoreceptors.There exist sensory cells with a dendrite penetrating one epidermis cell and bearing an embedded kinocilium and a collar of 8 stereocilia or ridges with a fribrillose substructure. These collared receptors probably function as mechanoreceptors.In comparison with collared sensory cells in species of other turbellarian orders, the embedded receptors in the Proseriata and Neorhabdocoela are more advanced and possess synapomorphous characteristics. With the embedded receptors a new evidence is given for the close phylogenetic relationship between the Proseriata and Neorhabdocoela.The distribution of collared cells in the animal system and their phylogenetic implication for a choanoflagellate origin of the Metazoa are briefly discussed.List of abbreviations ar annular rootlet - bm basement membrane - cb crystalline body - cc collar cell - cw cell web - cwt cell web-thickening - d dendrite - kc kinocilium - lm longitudinal musculature - mv microvilli - n nerve - nt neurotubuli - pb parenchymal branches - r rootlet - rd ridges - rh rhabdite - rm ring musculature - sc stereocilia - sd septate desmosomes - tm transversal musculature - u ultrarhabdites - za zonula adhaerens     

Plankton ecology in an ice-covered bay of Lake Michigan: utilization of a winter phytoplankton bloom by reproducing copepods

Plankton ecology was examined during the 1986 winter in Grand Traverse Bay, a 190 m deep, fjordlike bay on Lake Michigan. Before ice cover, algal concentration was low and uniformly distributed with depth, as it is in open Lake Michigan. During ice cover (February and March), a bloom of a typical winter-spring phytoplankton community developed in the upper 40 m, resulting in a 4 to 7-fold increase in feeding rate of adult Diaptomus spp. High algal concentration and zooplankton feeding persisted after ice melt (April). During and after ice cover, lipid concentrations of Diaptomus dropped rapidly from 34% of dry weight to 17 % because of egg production. High incident photosynthetically active radiation (PAR), high (45–50%) PAR transmittance of the ice due to little snow on the ice, and water column stability were probably responsible for the bloom. High ice transparency may be a common feature of large lakes and bays, where strong winds blow snow cover off the ice, or at low latitudes where snowmelt due to occasional rains and warm temperature is common. Winter reproducing calanoid copepods use these blooms to increase their reproductive output.