PubMed Health⌕ Search

Biomedical subjects

H Zwiers

Publications and source records attributed to H Zwiers.

At least 19 recordsLinked to original sources
PubMed1992

Peptides in the gastrointestinal tract in human immunodeficiency virus infection. The GI/HIV Study Group of the University of Calgary.

The presence of immunoreactivity to the neuronal phosphoprotein B-50 and the peptides bombesin, calcitonin gene-related peptide, galanin, neurotensin, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal polypeptide was examined in biopsy specimens from the duodenum and rectum of human immunodeficiency virus (HIV)-seropositive and HIV-seronegative male homosexual patients. The distribution of B-50 and the peptides was correlated with HIV serology, number of CD4+ lymphocytes, and the presence of HIV in biopsy culture. There was a very low incidence of enteric pathogens in both groups of patients. It was found that HIV-seropositive patients had a greater incidence of abnormal patterns of immunoreactivity (reduced intensity and/or density of innervation) in enteric nerves and enteroendocrine cells than HIV-seronegative patients. A reduction of substance P immunoreactivity was significantly correlated with reduced CD4+ lymphocyte count and HIV status; a similar trend was also seen for somatostatin and vasoactive intestinal polypeptide. Using B-50 as a marker, it was found that both groups of patients had altered patterns of immunoreactivity in rectal nerves. The findings of this study suggest that some of the clinical symptoms associated with HIV infection may be caused by a specific HIV enteropathy that influences enteric nerve and/or enteroendocrine cell function by altering the density of peptide immunoreactivity.

Adult↗
PubMed1991

B-50 (GAP-43): biochemistry and functional neurochemistry of a neuron-specific phosphoprotein.

The biochemistry and functional neurochemistry of the synaptosomal plasma membrane phosphoprotein B-50 (GAP-43) are reviewed. The protein is putatively involved in seemingly diverse functions within the nervous system, including neuronal development and regeneration, synaptic plasticity, and formation of memory and other higher cognitive behaviors. There is a considerable amount of information concerning the spatial and temporal localization of B-50 (GAP-43) in adult, fetal, and regenerating nervous tissue but far less is known about the physical chemistry and biochemistry of the protein. Still less information is available about posttranslational modifications of B-50 (GAP-43) that may be the basis of neurochemical mechanisms that could subsequently permit a variety of physiological functions. Hence, consideration is given to several plausible roles for B-50 (GAP-43) in vivo, which are discussed in the context of the cellular localization of the protein, significant posttranslational enzymes, and regulatory proteins, including protein kinases, phosphoinositides, calmodulin, and proteases.

Adrenocorticotropic Hormone↗
PubMed1990

Distribution of growth-associated protein, B-50 (GAP-43) in the mammalian enteric nervous system.

The presence of the growth-associated protein, B-50 (also known as GAP-43) was investigated in the adult mammalian enteric nervous system. The small intestine of rat, ferret and human was examined by immunohistochemistry. Dense B-50-like immunoreactivity was localized in nerves throughout the wall of the rat, ferret and human small intestine, notably in the myenteric and submucous plexuses, where in the ferret ileum it co-localized with vasoactive intestinal polypeptide-immunoreactive fibre groups. Material with the biochemical and immunological characteristics of rat B-50 was extracted from the rat ileum. In-situ hybridization demonstrated that enteric neurons express B-50. These findings are consistent with a role for B-50 in the documented plasticity of the adult enteric nervous system.

Animals↗
PubMed1990

Dephosphorylation of B-50 in synaptic plasma membranes.

Synaptic plasma membranes from rat brain cortex possess intrinsic ability to dephosphorylate the endogenous protein B-50. At low concentrations of [gamma-32P]ATP, B-50 phosphorylation in synaptic membranes is maximal at 30 seconds, followed by dephosphorylation for an additional 60 minutes. The dephosphorylation of 32P-labeled B-50 is not sensitive to the protease inhibitor leupeptin and not correlated with a loss of the B-50 content of synaptic membranes as measured with immunoblot analysis. Dephosphorylation of membrane-associated B-50 is stimulated to a small extent by Mg2+ but not by Ca2+. Heat-stable protein phosphatase inhibitors prevent dephosphorylation of 32P-labeled B-50. Dephosphorylation of B-50 in synaptic membranes is stimulated by ATP, ADP, or adenosine 5'-O-thiotriphosphate, but not by adenine, adenosine, other adenine or guanine nucleotides, nonhydrolyzable analogs of ATP or GTP, nor by adenosine 5'-O-(2-thiodiphosphate). B-50, phosphorylated by exogenous protein kinase C and purified to homogeneity, has been used as a substrate to follow the purification of B-50 phosphatase activity. B-50 phosphatase activity can be solubilized from synaptic membranes with 0.5% Triton X-100 and 75 mM KCl. Chromatography of the extract on DEAE-cellulose yields enhanced B-50 phosphatase activity.

Adenosine Triphosphate↗
PubMed1990

Binding of the neuronal protein B-50, but not the metabolite B-60, to calmodulin.

Protein kinase C phosphorylates the neurone-specific protein B-50 at a single Ser41 residue, which is also the point for a major proteolytic cleavage in vitro, and probably in vivo, that produces a B-50 phosphorylation-inhibiting N-terminal fragment and a large C-terminal metabolite B-60 (B-50(41-226]. The intact purified protein will bind to calmodulin in the absence of calcium, but the interaction has an absolute requirement for dephospho-B-50. In an attempt to unify two aspects of B-50 biochemistry, we have examined the interaction of B-50 binding to calmodulin and B-50 proteolysis. HPLC- and affinity-purified B-50 bound to calmodulin, but purified B-60 did not. To ensure that this effect was not due to the phosphorylation state of pure, isolated B-60, the metabolite was generated in vitro using a Triton extract of synaptosomal plasma membranes, which contains the as yet uncharacterized B-50 protease. B-60 derived from dephospho-B-50 also failed to bind calmodulin. The results demonstrate a direct connection between B-50 binding to calmodulin and B-50 proteolysis. The position of the proposed calmodulin-binding domain within intact B-50 is discussed in light of the failure of calmodulin to bind B-60.

Animals↗
PubMed1989

Evidence for a single protein kinase C-mediated phosphorylation site in rat brain protein B-50.

The neuronal protein B-50 may be involved in diverse functions including neural development, axonal regeneration, neural plasticity, and synaptic transmission. The rat B-50 sequence contains 226 amino acids which include 14 Ser and 14 Thr residues, all putative sites for phosphorylation by calcium/phospholipid-dependent protein kinase C (PKC). Phosphorylation of the protein appears to be a major factor in its biochemical and possibly its physiological activity. Therefore, we investigated rat B-50 phosphorylation and identified a single phosphorylated site at Ser41. Phosphoamino acid analysis eliminated the 14 Thr residues because only [32P]Ser was detected in an acid hydrolysate of [32P]B-50. Staphylococcus aureus protease peptide mapping produced a variety of radiolabelled [32P]B-50 products, none of which had the same molecular weights or HPLC retention times as several previously characterized fragments. Indirect confirmation of the results was provided by differential phosphorylation of major and minor forms of B-60 that have their N-termini at, or C-terminal to, the Ser41 residue and are the major products of specific B-50 proteolysis. Only those forms of B-60 that contained the Ser41 residue incorporated phosphate label. The results are discussed with reference to the substrate requirements for B-50 phosphorylation by PKC and the proposed structure of the B-50 calmodulin binding domain.

Amino Acid Sequence↗
PubMed1989

Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat.

Neurons that can regenerate their axons following axotomy increase their synthesis and axonal transport of a growth-associated protein, called GAP-43, which has been shown to be identical to the synaptic phosphoprotein B-50. The function of B-50/GAP-43 to the process of regeneration is unknown. We used a polyclonal, affinity-purified antibody against B-50 to study the axonal transport and localization of B-50/GAP-43-like immunoreactivity (B50LI) in the regenerating sciatic and facial nerves of adult rats. Quantitative data were obtained by densitometry of the B-50 band in immunoblots of nerve segments, which had been run on SDS-polyacrylamide gels. In the regenerating sciatic nerve, anterograde accumulation at a collection ligature was 3.0 times higher than retrograde accumulation. The mobile fraction of B50LI was only 0.28 of total B50LI and traveled with a mean anterograde velocity of 5.3 mm/hr. B50LI distribution in the newly regenerated portion of the nerve revealed maximal B50LI levels midway between the position of the crush and the fastest-growing axons. Immunocytochemistry of this portion of the nerve demonstrated B50LI to be associated with regenerating axons but also to a large extent with extra-axonal structures outlining the Schwann cell bands of Büngner. This zone of B50LI-positive Schwann cell bands was found to extend more distally in nerves in which regeneration had processed longer, e.g., up to 5 mm distal to the crush after 3 d and 8 mm after 4 d. Further distal to this zone, many fine regenerating axonal profiles could be detected with B-50 antibody, but were neurofilament negative. These findings raise the possibility of an extra-axonal function of B-50/GAP-43, as this protein might be secreted from regenerating axons and might play a role in axon-Schwann cell interactions during axonal maturation.

Animals↗
PubMed1988

Phosphorylation of the neuronal protein kinase C substrate B-50: in vitro assay conditions alter sensitivity to ACTH.

We have explored the hypothesis that changes in the in vitro assay conditions alter both the extent of endogenous phosphorylation of B-50 protein in synaptosomal plasma membrane (SPM) and also the ability of the neuropeptide, ACTH-(1-24) to inhibit the phosphorylation of this protein. B-50 phosphorylation is influenced by preincubation, pH and ionic strength. ACTH-(1-24)-induced inhibition of B-50 phosphorylation varies with ionic strength and SPM protein concentration. Reduction of the buffer ionic strength and the SPM protein concentration enhances the ability of ACTH-(1-24) to inhibit B-50 phosphorylation. Furthermore, loss of ACTH-(1-24) by adsorption to plastic pipettes and test tubes reduces the peptide concentration in the assay. Addition of a low concentration of bovine serum albumin (BSA) essentially eliminates this loss without affecting the extent of phosphate incorporation into B-50. These data provide an explanation for the relatively high (and variable) IC50 values for ACTH-(1-24)-induced inhibition of B-50 phosphorylation reported in the literature. Further, these data suggest that in vitro assay conditions must be carefully investigated before modulation of protein phosphorylation can adequately be studied.

Animals↗
PubMed1988

The growth-associated neuronal phosphoprotein B-50: improved purification, partial primary structure, and characterization and localization of proteolysis products.

A reversed phase HPLC procedure is reported that has allowed the separation of the growth-associated, kinase C substrate protein B-50 [previously purified by isoelectric focussing (IEF)] into three components (1-, m- and rB-50). The minor form 1B-50 (probably a proteolysis product) gave a 24-residue N-terminal amino acid sequence, but the major and possibly native form (mB-50) (and also rB-50 which is probably formed during IEF) appeared to be N-terminally blocked. HPLC also separated B-60, the major proteolysis product of B-50, into three components, and the N-terminal sequence of the major B-60 was determined. HPLC peptide mapping of SAP digests of the various B-50 and B-60 protein confirmed their close relationship, and four SAP generated fragments also afforded sequence data. The amino acid sequences obtained (1B-50, B-60 and fragments) are all found in the recently predicted (based on nucleotide sequencing) B-50/GAP43 sequence (226 amino acids), further confirming the identity of B-50 and GAP43, and helping clarify the relationship of B-60 (starting at position 41 of the predicted sequence) to B-50. Correlation of amino acid analyses, SAP fragment data, and the predicted sequence provided additional information on the length of the translated products, including evidence that the N-terminus of the major (blocked) form of B-50 starts at position 1 (Met) of the predicted sequence.

Animals↗
PubMed1988

Cloning and sequence analysis of cDNA for corticotropin-releasing factor precursor from the teleost fish Catostomus commersoni.

The sequence of a cDNA encoding the corticotropin-releasing factor precursor has been identified by screening lambda gt11 libraries constructed from poly(A)+ RNA of the hypothalamic region of the white sucker Catostomus commersoni brain with synthetic oligonucleotide probes deduced from the sequence of the rat corticotropin-releasing factor. The amino acid sequence of corticotropin-releasing factor of the sucker is strikingly conserved when compared to its counterpart from rat and differs only in two positions at the carboxyl terminus; in contrast, there is little similarity between their cryptic regions.

Amino Acid Sequence↗
PubMed1988

Anatomical distribution of the growth-associated protein GAP-43/B-50 in the adult rat brain.

GAP-43 (B-50,F1,pp46) is a neuron-specific phosphoprotein that has been implicated in the development and modulation of synaptic relationships. Although most neurons cease expressing high levels of GAP-43 after the completion of synaptogenesis (Jacobson et al., 1986), certain brain regions continue to have considerable amounts of the protein throughout life (Oestreicher et al., 1986); in at least one such area, the phosphorylation of the protein has been linked with the events that underlie synaptic potentiation (Lovinger et al., 1985). In this study, we used the indirect immunoperoxidase method to map the distribution of GAP-43/B-50 in the brains of 8 adult rats with 2 different antibodies: a monospecific, polyclonal antibody prepared in sheep against the purified protein and an affinity-purified IgG prepared in rabbits. Specific immunoreactivity was found primarily in the neuropil and followed a generally increasing caudal-to-rostral gradient along the neuraxis. Densest staining occurred in layer I of the cortex, the CA1 field of the hippocampus, and in a continuum of subcortical structures that included the caudate-putamen, olfactory tubercle, nucleus accumbens, bed nucleus of the stria terminalis, amygdala, and medial preoptic area-hypothalamus. In the brain stem, staining was seen in the central gray and in ascending visceral relay nuclei, but was essentially absent in areas related to ascending somatosensory information (e.g., the cochlear nuclei or vestibular complex) and motor control (e.g., nucleus ruber or the motor nuclei of the cranial nerves). Staining in dorsal thalamus was likewise modest in most somatosensory and somatomotor relay nuclei, but dark in certain other structures (e.g., mediodorsal nucleus, lateral complex). This distributional pattern raises the question of whether synapses in all areas containing high levels of GAP-43/B-50 are capable of undergoing functional plasticity, or whether the protein may function in some of these areas in some other capacity (e.g., general signal transduction).

Animals↗
PubMed1987

The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs.

The protein that has been designated as GAP-43, B-50, F1 or pp46 is associated with the growth and modulation of neuronal connections. cDNA clones for the rat and human genes were isolated and used to demonstrate that the messenger RNA for the protein is expressed only in neurons, that its overall level is highest in the developing brain, and that in the adult human brain levels of the mRNA are highest in the associative neocortex.

Animals↗
PubMed1986

Comparison of a 52-kDa phosphoprotein from synaptic plasma membranes related to long-term potentiation and the major coated vesicle phosphoprotein.

In the in vitro hippocampal slice preparation a short tetanus induces long-term potentiation (LTP) and an increase in the post hoc phosphorylation of a 52-kDa protein in synaptosomal plasma membranes (SPM) prepared from these slices. This 52-kDa SPM phosphoprotein closely resembles the predominant phosphoprotein in coated vesicles, pp50, with respect to the insensitivity of its phosphorylation to Ca2+/calmodulin and cyclic AMP. This resemblance prompted us to compare in rat brain the 52-kDa SPM protein with pp50 in isolated coated vesicles. Both proteins appear to be very similar on basis of the following criteria: relative molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, phospho-amino acid content, and isoelectric point. Since coated vesicles are thought to be involved in receptor-mediated endocytosis and membrane recycling, our data suggest that LTP-correlated changes in 52-kDa phosphorylation may reflect increased coated vesicle activity.

Action Potentials↗
PubMed1985

Modulation of the activity of purified phosphatidylinositol 4-phosphate kinase by phosphorylated and dephosphorylated B-50 protein.

To investigate the modulation of phosphatidylinositol 4-phosphate kinase activity by the degree of phosphorylation of the B-50 protein, the enzyme was purified from rat brain cytosol by ammonium sulphate precipitation and DEAE-cellulose column chromatography. Purified rat brain B-50 was phosphorylated with protein kinase C and dephosphorylated with alkaline phosphatase. Incubation of the semi-purified phosphatidylinositol 4-phosphate kinase with 1 microgram of the B-50 preparation enriched in the dephospho-form, resulted in a small reduction of phosphatidylinositol 4-phosphate kinase activity (-16%), whereas incubation with the phospho B-50 preparation inhibited the enzyme activity by 40%. The effect of exogenous B-50 was studied in the presence of 10 micrograms albumin to minimize aspecific protein-protein interactions. The present data on the effect of exogenous B-50 protein on phosphatidylinositol 4-phosphate kinase activity, further support our hypothesis that the phosphorylation state of B-50 may be a regulatory factor in phosphoinositide metabolism in rat brain.

Albumins↗
PubMed1985

Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.

This study describes the ultrastructural localization in rat hippocampal tissue in situ and in isolated synaptosomes of the brain-specific phosphoprotein B-50, using affinity purified anti-B-50 immunoglobulins (IgGs). Evidence is presented for the presynaptic localization of B-50 in rat brain. Given this specific localization a model is presented outlining the presumed function of the B-50 protein in the membrane and describing possible neuromodulation by adrenocorticotropin hormone (ACTH)-like peptides.

Animals↗
PubMed1985

Microdetermination of phosphoinositides in a single extract.

A method that allows the quantification of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-biphosphate (TPI) on a nanomolar scale is presented. The method is based on the simultaneous separation of lipids on high-performance thin-layer chromatography plates, followed by a microassay for phosphorus of PI spots and a densitometric assay of DPI and TPI. The new procedure allows the determination of the phospholipids in small amounts (100 micrograms protein) of synaptosomes and synaptic plasma membranes, and in homogenates of microwave-fixed brain tissue (1 mg wet wt). The usefulness of the method is illustrated by showing the effect of Ca2+ on the breakdown of DPI and TPI in synaptosomal plasma membranes.

Animals↗