PubMed Health⌕ Search

Biomedical subjects

L Baizer

Publications and source records attributed to L Baizer.

At least 19 recordsLinked to original sources
PubMed1996

Analysis of the role of calmodulin binding and sequestration in neuromodulin (GAP-43) function.

We demonstrated previously that forced expression of the neuronal phosphoprotein neuromodulin (also known as GAP-43, F1, B-50, and p57) in mouse anterior pituitary AtT-20 cells enhances depolarization-mediated secretion and alters cellular morphology. Here we analyze the role of calmodulin binding by neuromodulin in these responses. In cells expressing wild-type neuromodulin, a complex with calmodulin that is sensitive to intracellular calcium and phosphorylation is localized to the plasma membrane. Transfection of several mutant forms of neuromodulin shows that the effects of this protein on secretion are dependent on both calmodulin binding and association with the plasma membrane. In contrast, the morphological changes depend only on membrane association. Thus, the multitude of effects of neuromodulin noted in previous studies may result from divergent properties of this protein.

Animals↗
PubMed1996

Growth-associated protein-43 (GAP-43) facilitates peptide hormone secretion in mouse anterior pituitary AtT-20 cells.

The neuronal growth-associated protein (GAP)-43 (neuromodulin, B-50, F1), which is concentrated in the growth cones of elongating axons during neuronal development and in nerve terminals in restricted regions of the adult nervous system, has been implicated in the release of neurotransmitter. To study the role of GAP-43 in evoked secretion, we transfected mouse anterior pituitary AtT-20 cells with the rat GAP-43 cDNA and derived stably transfected cell lines. Depolarization-mediated beta-endorphin secretion was greatly enhanced in the GAP-43-expressing AtT-20 cells without a significant change in Ca2+ influx; in contrast, expression of GAP-43 did not alter corticotropin-releasing factor-evoked hormone secretion. The transfected cells also displayed a flattened morphology and extended processes when plated on laminin-coated substrates. These results suggest that AtT-20 cells are a useful model system for further investigations on the precise biological function(s) of GAP-43.

Animals↗
PubMed1995

Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy.

Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known.

Amino Acid Sequence↗
PubMed1995

Regulated expression of neurofibromin in migrating neural crest cells of avian embryos.

Neurofibromatosis type 1 (NF1) is a common human genetic disease involving various neural crest (NC)-derived cell types, in particular, Schwann cells and melanocytes. The gene responsible for NF1 encodes the protein neurofibromin, which contains a domain with amino acid sequence homology to the ras-guanosine triphosphatase activating protein, suggesting that neurofibromin may play a role in intracellular signaling pathways regulating cellular proliferation or differentiation, or both. To determine whether neurofibromin plays a role in NC cell development, we used antibodies raised against human neurofibromin fusion proteins in western blot and immunocytochemical studies of early avian embryos. These antibodies specifically recognized the 235 kD chicken neurofibromin protein, which was expressed in migrating trunk and cranial NC cells of early embryos (E1.5 to E2), as well as in endothelial and smooth muscle cells of blood vessels and in a subpopulation of non-NC-derived cells in the dermamyotome. At slightly later stages (E3 to E5), neurofibromin immunostaining was observed in various NC derivatives, including dorsal root ganglia and peripheral nerves, as well as non-NC-derived cell types, including heart, skeletal muscle, and kidney. At still later stages (E7 to E9), neurofibromin immunoreactivity was found in almost all tissues in vivo. To determine whether the levels of neurofibromin changed during melanocyte and Schwann cell development, tissue culture experiments were performed. Cultured NC cells were found to express neurofibromin at early time points in culture, but the levels of immunoreactivity decreased as the cells underwent pigmentation. Schwann cells, on the other hand, continued to express neurofibromin in culture. These data suggest, therefore, that neurofibromin may play a role in the development of both NC cells and a variety of non-NC-derived tissues.

Animals↗
PubMed1994

Purification and primary structure of Capl, an S-100-related calcium-binding protein isolated from bovine retina.

Calcium protein placental homolog (Capl) is an S-100-related calcium-binding protein selectively expressed in cell lines that have been induced to grow or differentiate. In addition, the expression of Capl correlates with the induction of the metastatic phenotype in tumor cell lines and the transformation of normal cells by activated oncogenes or chemical carcinogens. Although not previously associated with the nervous system, in this study, Capl was purified from bovine neural retina by a combination of phenyl-Sepharose and organomercurial chromatography. The complete amino acid sequence of bovine Capl was established primarily by Edman degradation of peptides generated by cleavage of methionyl, lysyl, glutamyl, and aspartyl bonds. NH2-terminal methionyl and aspartyl peptides were analyzed by tandem mass spectrometry, which provided the sequence of the first 8 residues and identified the NH2-terminal blocking group as an acetyl moiety. The molecular mass of the intact protein determined by electrospray mass spectrometry (M(r) = 11,716.75 +/- 0.42) and the calculated molecular mass deduced from the amino acid composition (M(r) = 11,718) were in agreement, thus supporting the accuracy of the sequence assignment. Capl isolated from the retina was shown to be indistinguishable by mass and immunochemical properties from its counterpart in the bovine aorta, which previously was the only source of purified Capl. Northern analysis using cloned Capl cDNA revealed that Capl mRNA is present not only in the retina but the choroid as well. Further support for choroidal localization came from immunohistochemical experiments using specific anti-Capl antibodies. The physiological significance of Capl in ocular tissues and the aorta is discussed.

Amino Acid Sequence↗
PubMed1993

Analysis of the sequence and embryonic expression of chicken neurofibromin mRNA.

Neurofibromatosis type 1 (NF1) is a common inherited disorder that primarily affects tissues derived from the neural crest. Recent identification and characterization of the human NF1 gene has revealed that it encodes a protein (now called neurofibromin) that is similar in sequence to the ras-GTPase activator protein (or ras-GAP), suggesting that neurofibromin may be a component of cellular signal transduction pathways regulating cellular proliferation and/or differentiation. To initiate investigations on the role of the NF1 gene product in embryonic development, we have isolated a partial cDNA for chicken neurofibromin. Sequence analysis reveals that the predicted amino acid sequence is highly conserved between chick and human. The chicken cDNA hybridizes to a 12.5-kb transcript on RNA blots, a mol wt similar to that reported for the human and murine mRNAs. Ribonuclease protection assays indicate that NF1 mRNA is expressed in a variety of tissues in the chick embryo; this is confirmed by in situ hybridization analysis. NF1 mRNA expression is detectable as early as embryonic stage 18 in the neural plate. This pattern of expression may suggest a role for neurofibromin during normal development, including that of the nervous system.

Amino Acid Sequence↗
PubMed1993

Regulated expression of the neurofibromin type I transcript in the developing chicken brain.

Neurofibromatosis type 1 (NF-1) is among the most common inherited diseases affecting cells of the central and peripheral nervous systems. A region of the NF-1 gene is similar in sequence to the ras-GTPase activator protein (ras-GAP), and investigations have confirmed that the NF-1 gene product (now known as neurofibromin) stimulates ras-GTPase activity in vitro and in vivo. Neurofibromin modulates the ability of ras proteins to regulate cellular proliferation and/or differentiation, suggesting a possible role in normal development. An alternative form of the neurofibromin transcript with an additional 63-bp exon inserted in the GAP-related domain (GRD) has been described recently. To determine whether differential expression of the two forms of neurofibromin GRD mRNA plays a role in embryonic development, we have isolated and characterized the corresponding chicken cDNA. The predicted amino acid sequence for the inserted exon is identical between chick and human, as are the exon-intron boundaries. RNase protection and RNA-polymerase chain reaction analyses demonstrate that most tissues express predominantly type II mRNA (which contains the insert) throughout embryonic development. In contrast, whereas type II is the major form in the brain early in development, expression of the type I transcript (without the insert) in this tissue increases dramatically at later times. Analysis of primary cultures derived from chick embryo brain indicates that the type I mRNA is enriched in neurons.

Amino Acid Sequence↗
PubMed1993

Transient expression of GAP-43 in nonneuronal cells of the embryonic chick limb.

Growth-associated protein (GAP)-43 is highly expressed in neuronal growth cones during periods of axonal outgrowth in development and regeneration of the nervous system. Although GAP-43 is generally considered to be neuron-specific, it is also expressed in some glial cells of the peripheral and central nervous systems and in at least two populations of mesodermally-derived cells in the developing chick limb. GAP-43 mRNA is expressed transiently in developing limbs; although this expression is correlated temporally with the ingrowth of neurites and axons to the limbs, it appears to be independent of nerves. Immunoreactivity for GAP-43 colocalizes in some developing limb muscle and GAP-43 mRNA and protein are particularly abundant in the interdigital mesenchyme that undergoes programmed cell death. GAP-43 has been postulated to mediate rapid changes in cell shape in neurons and glial cells and may serve a similar function in myoblasts fusing to form myotubes and in apototic and phagocytic cells of the interdigital mesenchyme.

Animals↗
PubMed1992

Transient expression of GAP-43 in nonneuronal cells of the embryonic chicken limb.

Growth associated protein (GAP)-43 is a membrane-bound phosphoprotein expressed in neurons and is particularly abundant during periods of axonal outgrowth in development and regeneration of the nervous system. In previous work, we cloned a full-length chicken GAP-43 cDNA and described the expression of its corresponding mRNA during early development of the chicken nervous system. We report here that the GAP-43 mRNA is also expressed transiently in developing limbs of chicken embryos, which contain axons of spinal cord and dorsal root ganglion neurons, but do not contain neuronal cell bodies. GAP-43 mRNA was first detectable by RNA blot analysis in limbs from Embryonic Day 5 (E5) embryos, reached maximal levels between E6 and E8, and diminished by E10. In situ hybridization analysis showed that the GAP-43 mRNA was localized in distal regions of developing limbs and was particularly abundant in the mesenchyme surrounding the digital cartilage. In some regions of the limb, GAP-43 immunoreactivity colocalized in cells that were also immunoreactive for meromyosin, a muscle-specific marker. These data suggest that both GAP-43 mRNA and the protein are expressed in nonneuronal cells of the developing limb, some of which may be part of the muscle cell lineage.

Animals↗
PubMed1992

GAP-43 in non-neuronal cells of the embryonic chick limb: clues to function.

The expression of GAP-43 in developing and regenerating neurons has been well characterized, but the function of this membrane-bound phosphoprotein is still unclear. Although GAP-43 is considered to be neuron-specific, it is also expressed in various glial cells of the peripheral and central nervous systems and in at least two populations of mesenchymal cells in the developing chick limb. GAP-43 mRNA is expressed transiently in developing limbs, which contain axons of spinal cord and dorsal root ganglion neurons, but do not contain neuronal cell bodies. This expression is correlated temporally with the in-growth of neurites and axons to the limbs, but appears to be independent of nerves. In some regions of the limb, GAP-43 immunoreactivity co-localizes in cells that are also immunoreactive for meromyosin, a muscle-specific marker. In addition, GAP-43 mRNA and protein are particularly abundant in the interdigital mesenchyme that undergoes apoptosis, or programmed cell death. GAP-43 has been postulated to mediate rapid changes in cell shape and the extension of processes in neuronal growth cones and elongating axons. We suggest here that GAP-43 may serve a similar function in glial cells, in myoblasts fusing to form myotubes, and in apoptotic and phagocytic cells of the interdigital mesenchyme.

Animals↗
PubMed1990

Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.

Amino Acid Sequence↗
PubMed1990

Chicken growth-associated protein (GAP)-43: primary structure and regulated expression of mRNA during embryogenesis.

Growth-associated protein (GAP)-43 is a neuron-specific phosphoprotein whose expression is associated with axonal outgrowth during neuronal development and regeneration. In order to investigate the expression of this gene product in the early developing nervous system we have isolated and sequenced a cDNA for chicken GAP-43. The predicted amino acid sequence for chicken GAP-43 displays extensive similarity to that of the mammalian protein, particularly in the amino-terminal region, to which functional domains of the protein have been assigned. The cDNA hybridizes with two RNAs of differing molecular weights on Northern blots; both appear to be regulated similarly. These RNAs first appear in the brain on embryonic day 3 (E3), suggesting that GAP-43 begins to be expressed when neuroblasts become post-mitotic. In situ hybridization analysis reveals that GAP-43 RNA is expressed by several neural structures in the chick embryo, including derivatives of the neural tube, neural crest, and neuroectodermal placodes.

Amino Acid Sequence↗
PubMed1990

Chicken growth-associated protein GAP-43 is tightly bound to the actin-rich neuronal membrane skeleton.

We have identified the chicken equivalent of growth-associated protein GAP-43 in a detergent-resistant membrane skeleton from cultures of chick neurones and embryonic chick brain. Antisera to the membrane skeleton protein, the 3D5 antigen, precipitate the translation product of chick GAP-43 cDNA, and the 3D5 antigen is also detected by antisera against synthetic peptides from the known amino acid sequence of rat GAP-43. The chick protein and the rat GAP-43 are biochemically similar proteins that both serve as major targets of phosphorylation by endogenous protein kinase C. The detergent-resistant complex in which GAP-43 is found also contains actin (approximately 5% of the total protein) and a neurone-specific cell surface glycoprotein. We suggest that the membrane skeleton of neurones may be a primary site of action of GAP-43.

Actins↗
PubMed1987

Recognition of specific targets by cultured dorsal root ganglion neurons.

We have assessed the effects of different target cell populations on axonally transported proteins by the use of compartmental cell culture systems that separate the soma from the growing axons of rat sensory neurons. The labeling of 3 rapidly transported proteins diminishes when the growing axon contacts spinal cord cells (which are normal in vivo targets), and remains unaffected by contact with fibroblasts or heart cells. Medium conditioned by spinal cord cells does not exert this effect. Thus, specific classes of cells may be distinguished as target tissue by sensory neurons in vitro. Such recognition is accompanied by specific molecular changes in axonally transported proteins.

Animals↗
PubMed1985

Neuronal traits of clonal cell lines derived by fusion of dorsal root ganglia neurons with neuroblastoma cells.

In an attempt to immortalize the gene products of single neurons, somatic cell hybrids were produced by fusion of embryonic rat dorsal root ganglion (DRG) neurons with mouse neuroblastoma cells. Embryonic day 13 rat DRGs were fused with mouse neuroblastoma cells deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8). The hybrid cells were selected in medium with 100 microM hypoxanthine/1 microM aminopterin/12 microM thymidine to eliminate the neuroblastoma cells and with cis-hydroxyproline to retard fibroblast growth. Of the 17 lines derived, 4 manifested neuronal properties and were cloned. These lines retain both rat and mouse chromosomes and synthesize characteristic rat and mouse isoenzymes. Neuronal gangliosides, action potentials, and extensive neurite-like processes are exhibited by these hybrid cells, properties characteristic of DRG neurons but not of the neuroblastoma parent. Each line manifests a unique combination of action-potential properties and cell-surface markers, suggesting the selective expression of subsets of DRG neuronal genes. All of these neuronal properties are expressed constitutively, without the need for chemical induction or mitotic inhibition, and stably, without diminution after at least 5 months in culture. These lines may prove useful in the identification and isolation of gene products that characterize individual or small subsets of DRG neurons.

Animals↗
PubMed1985

Regulation of dopamine release from PC12 pheochromocytoma cell cultures during stimulation with elevated potassium or carbachol.

To examine the role of cyclic AMP in the process of catecholamine release experiments have been performed with cultures of PC12 pheochromocytoma cells. Elevated potassium (56 mM) and carbamylcholine (carbachol, 10(-4) M) cause rapid increases in cyclic AMP levels in the cultures that show a time course similar to that of evoked dopamine release. These secretogogue-induced increases in cyclic AMP levels are well correlated with release in terms of relative magnitude and calcium dependence. Forskolin (a direct activator of adenylate cyclase) causes dose-related increases in cyclic AMP levels in PC12 cell cultures that are synergistic with those caused by either elevated potassium or carbachol. At low concentrations forskolin significantly increases evoked release, whereas at higher concentrations it increases both spontaneous and evoked release. These results suggest that cyclic AMP may be involved in the process of dopamine release from PC12 cells in culture.

Animals↗
PubMed1985

Nerve growth factor treatment enhances nicotine-stimulated dopamine release and increases in cyclic adenosine 3':5'-monophosphate levels in PC12 cell cultures.

In order to examine the relationship between cyclic AMP (cAMP) levels and evoked neurotransmitter release, experiments have been performed with cultures of clonal rat PC12 pheochromocytoma cells. Stimulation of the release of endogenous dopamine by nicotine in these cultures is calcium-dependent and blocked by d-tubocurarine, a specific nicotinic cholinergic antagonist. Similarly, nicotine causes increases in cAMP levels in PC12 cell cultures that are calcium-dependent and blocked by d-tubocurarine. Cultures treated for 6 days or longer with 2 X 10(-9) M nerve growth factor (NGF) release a 3- to 4-fold greater amount of dopamine than do control cultures in response to a maximal concentration of nicotine. Correspondingly, nicotine causes a 3-fold greater increase in cAMP levels in the NGF-treated cultures than in the controls. These results suggest that stimulation of the nicotinic cholinergic receptor in PC12 cells results in some manner in the activation of adenylate cyclase and further support the notion that cAMP is involved in the process of neurotransmitter release.

Animals↗