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At least 1,261 records · Page 70Linked to original sources
PubMed1990

Changes in the distribution of GAP-43 during the development of neuronal polarity.

GAP-43, a neuron specific growth-associated protein, is selectively distributed to the axonal domain in developing neurons; it is absent from dendrites and their growth cones. Using immunofluorescence microscopy, we have further examined the distribution of GAP-43 during the development of hippocampal neurons in culture, in order to determine when this polarized distribution arises. Cultured hippocampal neurons initially extend several short processes which have the potential to become either axons or dendrites. At this stage, before the morphological expression of polarity, GAP-43 is concentrated in the growth cones of these processes but is distributed more or less equally among them. Polarity becomes established when one of these processes elongates to become the axon. At the earliest stage when the emerging axon can be identified, GAP-43 is preferentially concentrated in its growth cone. During the next few days, as the remaining processes take on dendritic properties, they lose their residual GAP-43 immunoreactivity. Throughout development, GAP-43 remains highly concentrated in the axonal growth cone, but the concentration of GAP-43 in the axon shaft increases, beginning near the growth cone and progressing proximally until GAP-43 is uniformly distributed along the entire axon. At all stages of development, GAP-43 is also concentrated in the region of the Golgi apparatus. These results suggest that the selective sorting of at least one membrane protein into the axon coincides with the morphological expression of polarity. These results also raise the possibility that GAP-43 may play an important role in the early phases of axonal outgrowth, by which the functional polarity of neurons is established.

Animals↗
PubMed1987

[Serotyping Neisseria gonorrhoeae].

To control gonorrhea an understanding of pathogenesis and epidemiology is necessary. To achieve this goal a serologic typing system recognizing antigenic variants of the gonococcus is needed. The serotyping systems proposed in past years are described and their clinical relevance discussed. At the moment two typing systems based on protein I and monoclonal antibodies are available. Protein I serotypes are correlated with the killing of Neisseria gonorrhoeae by bactericidal antibodies and may play a role in the future development of a gonococcal vaccine. The serovar system permits precise analysis of gonococcal populations and can be used especially for epidemiologic studies. The possibility of discriminating between reinfection and therapy failure can be of importance in therapeutic studies. A microepidemiologic analysis for partner identification is possible by combined determination of serovar and auxotype. Both monoclonal typing systems represent standardized monospecific reagents which, besides serotyping, will be used for the diagnosis of gonococcal disease as well.

Antibodies, Monoclonal↗
PubMed1989

Functional analysis of Ca2+/calmodulin-dependent protein kinase II expressed in bacteria.

The cDNA encoding the 50-kDa subunit of Ca2+/calmodulin (CaM)-dependent protein kinase II from adult rat brain was cloned into the bacterial expression vector pK223-2 and produced in bacteria. Extensive modification of the cDNA was required to express detectable levels of enzyme. The activity of the bacterially expressed kinase was stringently dependent on Ca2+/CaM but did not exhibit cooperative activation kinetics characteristic of the forebrain enzyme and required 10-fold greater amounts of CaM for half-maximal activation. The bacterially expressed enzyme displayed an apparent Km for a synthetic peptide substrate similar to that of the forebrain enzyme (12 and 10 microM, respectively). Limited proteolysis maps of autophosphorylated peptides, and Western blot analysis demonstrated that the bacterially expressed enzyme was structurally and immunologically indistinguishable from the 50-kDa subunit of the rat forebrain holoenzyme. The bacterially expressed enzyme became Ca2+/CaM-independent after Ca2+/CaM-dependent autophosphorylation in a fashion identical to the forebrain enzyme.

Animals↗
PubMed1989

[Immunobiologic aspects of Neisseria gonorrhoeae].

Surface structures of Neisseria gonorrhoeae (NG), which can mediate properties of virulence like adhesion and inflammation, try to escape the human immune response with the help of their highly variable antigenicity. In addition there are surface-exposed structures, like protein 1, which have relatively constant antigenetical powers and are likely to play an important part in both the uptake of nutrients and the infection process. Protein 1 shows no intrastrain and only limited interstrain variability. Anti-protein 1 monoclonal antibodies have gained practical significance with regard to serotyping and detection of NG in clinical specimens. Conserved epitopes of pilin and protein 1 may serve as the basis of a gonococcal vaccine.

Antibodies, Monoclonal↗
PubMed1989

Localization of 4.1 related proteins in cerebellar neurons.

Localization of 4.1 related proteins in neurons was studied with immunofluorescence microscopy and with immunoelectron microscopy on ultrathin cryosections. In rat cerebellum, 4.1 immunoreactive proteins were demonstrated in Purkinje cell bodies, dendrites and other neurons in the cerebellar cortex. Some glial cells showed staining, but no labeling was found in myelinated axons of the white matter and of the glomeruli in the granule cell layer. At the ultrastructural level, the 4.1 related proteins were localized mainly in the cytoplasmic matrix, while some labeling was found underneath the plasma membrane. To determine whether 4.1 related proteins in neuronal cytoplasm exist as part of the cytoskeleton or not, PC12 cells cultured in the presence of nerve growth factor were stained with the anti-4.1 antibody. Since cytoplasmic staining was retained after detergent treatment, the 4.1 related proteins seem to exist as a component of the neural cell cytoskeleton. Localization of 4.1 related proteins during the postnatal development of the cerebellum was also studied. In Purkinje cells, localization of 4.1 related proteins changed according to the stages of the postnatal development. The present data suggest that 4.1 related proteins in neurons localized mainly in the cytoplasm and may play some role in organizing cytoskeletal networks in the cytomatrix. Their distribution is developmentally regulated in some neurons, possibly in relationship to their maturation in the cytoskeleton.

Animals↗
PubMed1988

Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation.

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.

Benzofurans↗
PubMed1986

Acute ACTH regulation of adrenal corticosteroid biosynthesis. Rapid accumulation of a phosphoprotein.

Two-dimensional gel electrophoresis was used to monitor proteins synthesized in unstimulated control and in adrenocorticotropic hormone (ACTH)- or cAMP-stimulated rat adrenal cells. Four proteins, which have similar proteolytic peptide maps, have been identified. The two found primarily in unstimulated cells are referred to as pb and pa, where pb is the protein with more basic isoelectric point. Similarly, proteins ib and ia were detected only in stimulated cells. The synthesis of pb occurs only in unstimulated cells and that of ib only in stimulated cells. Protein ib accumulates with the same lag time, rate, and stimulant dose response as the increase in steroid hormone synthesis. Pulse-chase studies showed that protein ib is not produced from pb by a post-translational modification. Proteins pb and ib thus seem identical with proteins p and i previously identified in rat adrenal cortex and corpus luteum (Krueger, R.J., and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167, and Pon, L.A., and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6594-6599). The acidic forms, pa and ia, appear after a longer lag time and are produced at a slower rate than the basic forms. Pulse-chase studies showed that the disappearance of the basic form of each protein occurs concurrently with the appearance of the corresponding acidic form. Addition of [32P]orthophosphate to stimulated adrenal cells allowed direct demonstration that proteins ib and ia are phosphorylated. Moreover, alkaline phosphatase treatment of [35S]methionine-labeled, cAMP-stimulated adrenal cells caused a large decrease in the amounts of ib and ia and the appearance of proteins with the same two-dimensional electrophoretic mobilities as pb and pa. These observations suggest that protein ib may mediate stimulation of steroidogenesis, be produced by an ACTH- or cAMP-dependent, cotranslational phosphorylation of protein pb, and be lost by a cycloheximide-insensitive, post-translational conversion to ia.

Adrenal Cortex Hormones↗
PubMed1987

Similarities between protein IIIa and protein IIIb, two prominent synaptic vesicle-associated phosphoproteins.

Protein IIIa (Mr 74,000) and protein IIIb (Mr 55,000) are 2 major phosphoproteins found in mammalian brain. It was previously shown in intact nerve cells that the phosphorylation state of these 2 proteins could be increased by electrical stimulation, by depolarizing agents in the presence of calcium, and by 8-bromo-cAMP. We now report that protein IIIa and protein IIIb possess significant structural homology, as indicated by immunochemical studies using polyclonal and monoclonal antibodies and by peptide-mapping studies. A quantitative radioimmunoassay using immunolabeling in SDS-polyacrylamide gels has been used to study the tissue distribution and regional and subcellular distribution in the brain of the 2 proteins. The 2 proteins were found only in nervous tissue and the adrenal medulla. Subcellular fractionation studies suggested that both proteins are associated with synaptic vesicles.

Animals↗
PubMed1988

Cholinergic regulation of protein III phosphorylation in bovine adrenal chromaffin cells.

Protein IIIa (Mr approximately 74,000) and protein IIIb (Mr approximately 55,000), referred to collectively as protein III, are synaptic vesicle-associated phosphoproteins found in all regions of the rat nervous system and in the rat adrenal medulla. In the present study, the presence of protein III and the regulation of its phosphorylation were examined in chromaffin cells isolated from bovine adrenal medullae. Protein III was present in chromaffin cells isolated and purified from bovine adrenal medullae. The levels of protein III were moderately enriched in purified chromaffin cells compared with whole adrenal medullae. Preincubation of chromaffin cells with 32PO4 led to the endogenous phosphorylation of protein III, and phosphopeptide maps of chromaffin cell protein III were similar to those of protein III from bovine cerebral cortex. Treatment of the chromaffin cells with ACh produced calcium-dependent increases in both the phosphorylation of protein III and the release of 3H-norepinephrine. These effects of ACh were mimicked by nicotine but not by muscarine. Other secretagogues (elevated K+, veratridine, Ba2+) also increased both the phosphorylation of protein III and the release of 3H-norepinephrine. However, detailed characterization of the secretagogue-induced increases in protein III phosphorylation and 3H-norepinephrine secretion suggested that protein III phosphorylation was more directly associated with an increase in intracellular calcium than with secretion per se.

Acetylcholine↗
PubMed1983

Adenosine triphosphate-dependent uptake of glutamate into protein I-associated synaptic vesicles.

Protein I is a neuron-specific, synaptic phosphoprotein highly localized on the surface of synaptic vesicles. We have recently isolated anti-Protein I IgG by affinity chromatography and shown that these antibodies inhibit specifically the phosphorylation of Protein I (Naito, S., and Ueda, T. (1981) J. Biol. Chem. 256, 10657-10663). In an effort to characterize Protein I-associated synaptic vesicles with respect to the types of neurotransmitters, we have now developed a procedure, using the affinity-purified anti-Protein I IgG, which allows immunoprecipitation of those synaptic vesicles which contain Protein I. The isolated vesicles are largely free of contamination from other intracellular organelles and plasma membranes. We present evidence that these vesicles isolated from bovine cortex are able to accumulate L-glutamate specifically in an ATP-dependent, temperature-dependent but Na-independent manner. Thus, the structurally similar aminoacid neurotransmitters aspartate and gamma-aminobutyric acid, as well as other neurotransmitters such as dopamine, norepinephrine, serotonin, acetylcholine, and glycine, failed to show a significant ATP-dependent uptake into these vesicles. Moreover, the ATP-dependent glutamate uptake was not inhibited effectively by glutamine, aspartate, or gamma-aminobutyric acid. The ATP-dependent glutamate uptake requires ATP hydrolysis; thus there was little accumulation of glutamate in the absence of ATP or Mg2+, or when ATP was replaced by an unhydrolyzable beta, gamma-methylene ATP analog. The glutamate uptake appears to be driven at least in part by a membrane potential generated by Mg2+-ATPase, similar to that of the catecholamine and serotonin uptakes into storage granules. These observations suggest that Protein I may be involved in some aspect of the function of glutamate-containing synaptic vesicles in the brain.

Adenosine Triphosphate↗
PubMed1981

Neurotransmitter- and neuromodulator-dependent alterations in phosphorylation of protein I in slices of rat facial nucleus.

Protein I is a neuronal phosphoprotein associated primarily with synaptic vesicles. Regulation of its state of phosphorylation has been investigated in slices of rat facial nucleus. This brainstem motor nucleus has a facilitatory serotonergic input and contains no interneurons. Serotonin (5-hydroxytryptamine, 5-HT, 10(-4) M), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 4 x 10(-5) M), converted approximately 26% of Protein I in these slices from the dephospho-form to the phospho-form. This effect was partially inhibited using two classical 5-HT antagonists, mianserin added to the slices during in vitro incubation and metergoline administered in vivo. The effect of 5-HT appeared to be Ca2+-dependent, unlike that of IBMX (10(-3) M). Adenosine, its analog 2-chloroadenosine, and ATP also increased the phosphorylation of Protein I in facial nucleus slices. 2-Chloroadenosine (5 x 10(-4) M) caused a 29% phosphorylation of Protein I, and this effect was not dependent on extracellular Ca2+. The phosphorylation of Protein I caused both by 2-chloroadenosine and by ATP was inhibited by the adenosine antagonist 2'-deoxyadenosine. Results of additional experiments suggest that the great majority of the Protein I in the facial nucleus is present in presynaptic terminals other than the serotonergic afferents. It is concluded that the stimulation by 5-HT and adenosine of Protein I phosphorylation results largely from a direct action of these compounds on those Protein I-containing terminals.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases.

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.

Animals↗