Biomedical subjects

K Akagawa

Publications and source records attributed to K Akagawa.

At least 37 records · Page 2Linked to original sources

PubMed1997

Differential expression of neuroD in primary cultures of cerebral cortical neurons.

We have investigated the expression patterns of a basic helix-loop-helix regulatory gene, neuroD, in primary cultures of murine cerebral cortical neurons. The differentiation states of neurons in primary cultures were determined by the sensitivity of neurons to glutamate toxicity and the expression of specific proteins such as the phosphorylated form of a 200-kDa neurofilament, HPC-1/syntaxin 1A, and cell adhesion molecule L1. The expression of neuroD was determined by RT-PCR analysis and in situ hybridization. The experimental results thus obtained revealed that neuronal maturation is initiated between Day 7 and Day 11 in the culture as already known, and that the expression of neuroD decreases with increasing days in culture. Based on these findings, it was concluded that neuroD is expressed in immature neurons but not in mature ones.

Animals↗

PubMed1997

Differential involvement of synaptic vesicle and presynaptic plasma membrane proteins in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by progressive cognitive decline. Recent studies have shown that synaptic loss in the cortex is the major correlate of cognitive decline in AD. In the present study we assessed synaptic proteins such as synaptobrevin, synaptophysin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), and syntaxin1/HPC-1 in control and AD brains to determine whether synaptic proteins are equally or differentially affected in AD. Western analysis showed that in AD levels of synaptobrevin and synaptophysin were decreased by some 30% from amounts in controls, while those of synaptotagmin, SNAP-25, and syntaxin 1/HPC-1 were decreased by only about 10%. As synaptobrevin and synaptophysin are localized mainly in transmitter-containing synaptic vesicles while SNAP-25 and syntaxin 1/HPC-1 are found in presynaptic plasma membranes, these results suggest differential involvement of synaptic components in AD.

Alzheimer Disease↗

PubMed1997

Mapping of the human HPC-1/syntaxin 1A gene (STX1A) to chromosome 7 band q11.2.

We previously described the cDNA sequence of HPC-1/syntaxin 1A (HGMW-approved symbol STX1A) from rat and bovine brains. HPC-1/syntaxin 1A belongs to the syntaxin family and is apparently involved in intracellular membrane transport and the exocytosis of neurotransmitters. In this study, we isolated the cDNA and the genomic DNA clone for human HPC-1/syntaxin 1A and carried out gene mapping. Polymerase chain reaction analysis of human/rodent somatic cell hybrid panels and fluorescence in situ hybridization analysis using a genomic DNA clone provided evidence that the gene for human HPC-1/syntaxin 1A maps to chromosome region 7q11.2.

Animals↗

PubMed1997

Interaction of HPC-1/syntaxin 1A with the cytoskeletal protein, tubulin.

It is believed that HPC-1/syntaxin 1A regulates the intracellular membrane transport. We found a possible tubulin binding motif like sequence in residues 89 approximately 106 of HPC-1/syntaxin 1A. To determine whether or not HPC-1/syntaxin 1A binds to tubulin, we performed in vitro binding studies. We showed that both the rat brain and recombinant HPC-1/syntaxin 1A bound to tubulin in vitro. Competition experiments with synthetic peptides revealed that HPC-1/syntaxin 1A bound to tubulin at residues 89 approximately 106 which were supposed to constitute the tubulin binding consensus sequence.

Amino Acid Sequence↗

PubMed1997

Overexpressed syntaxin 1A/HPC-1 inhibits insulin secretion via a regulated pathway, but does not influence glucose metabolism and intracellular Ca2+ in insulinoma cell line beta TC3 cells.

We have previously established a stable beta TC3 cell line that overexpresses syntaxin 1A, designated beta TC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using beta TC-hpc1 cells, we aimed to determine whether syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin. beta TC3 and beta TC-hpc1 cells were simultaneously pulse-labeled with 3H-leucine for 30 min in 11 mM glucose and chased for 1 h in one of a number of different concentrations of TPA in 11 mM glucose. Total immunoreactive insulin release (IRI) by both cell types during the chase period was markedly increased by the addition of TPA in a dose-dependent manner; however, the IRI from beta TC-hpc1 cells was lower than that from beta TC3 cells. The secretion of newly synthesized proinsulin/insulin from both cell types, which in beta TC3 cells is thought to occur via a constitutive pathway, was in the same range under any condition. Thus, the evidence indicates that syntaxin 1A preferentially functions in the regulated insulin release pathway in beta TC3 cells. In order to clarify the effect of overexpressed syntaxin 1A on glucose metabolism and intracellular Ca2+ we analyzed the glucose transport system, glucose phosphorylation activity, and cytosolic concentration of free Ca2+ ([Ca2+]i). 2-Deoxy-glucose uptake and the content of GLUT1 protein in the plasma membrane fractions of beta TC-hpc1 cells were not different from those of beta TC3 cells. Radiometric assays of glucose phosphorylation activity showed that there were no differences in hexokinase activity and glucokinase activity between beta TC3 and beta TC-hpc1 cells. [Ca2+]i measured by using fura 2 demonstrated that there was no difference in [Ca2+]i between beta TC3 and beta TC-hpc 1 cells under glucose-stimulated conditions. The present experiments indicate that syntaxin 1A plays a central role in a late step of the regulatory insulin release pathway without a change in glucose metabolism and [Ca2+]i in beta TC3 cells.

Animals↗

PubMed1997

Characterization of HPC-1 antigen, an isoform of syntaxin-1, with the isoform-specific monoclonal antibody, 14D8.

We raised polyclonal and monoclonal antibodies against rat recombinant HPC-1/syntaxin 1A lacking a transmembrane domain. The polyclonal antibody recognized two major bands at 35 and 40 kDa from rat brain membranes. A hybridoma clone designated 14D8, however, recognized only one band at 35 kDa. A polyclonal antibody detected recombinant syntaxin 1B, as well as HPC-1/syntaxin 1A on an immunoblot, whereas 14D8 recognized recombinant HPC-1/ syntaxin 1A, but not syntaxin 1B. Therefore, 14D8 is specific for HPC-1/syntaxin 1A. Using this monoclonal antibody, we investigated the expression of HPC-1/syntaxin 1A in the rat hippocampal membranes. HPC-1/syntaxin 1A was present even in the embryonic d 19 (E19) hippocampal membranes, and it increased during the next two postnatal wk. Pyramidal cell axons were intensely stained with the 14D8 monoclonal antibody, suggesting that HPC-1/syntaxin 1A was not restricted to the presynaptic terminal. Furthermore, we investigated the phosphorylation of HPC-1/syntaxin 1A in the rat brain membranes. HPC-1/syntaxin 1A affinity-purified on a 14D8 IgG-coupled column was recognized by antiphosphoserine antibody, but not by antiphosphotyrosine and phosphothreonine antibodies.

Animals↗

PubMed1997

Transient decrease of HPC-1/syntaxin-1A mRNA in the rat hippocampus by kainic acid.

HPC-1/syntaxin-1A is a neuronal protein of which the mRNA has an immediate early gene-like structure in its 3'-untranslated region. Whereas HPC-1/syntaxin-1A protein plays a crucial role in neurotransmitter release, little is known about HPC-1 gene expression. We demonstrate here that HPC-1 mRNA expression in rat hippocampal neurons in vivo decreased 8 h after kainic acid (KA) administration, but was restored thereafter. The transient decrease of HPC-1 mRNA upon KA administration suggests that the HPC-1 mRNA expression in neurons could be altered by excitation by trans-synaptic stimulation.

Animals↗

PubMed1997

Monocyte-derived dendritic cells represent a transient stage of differentiation in the myeloid lineage.

Cultivation of human peripheral blood monocytes with granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-4 facilitates generation of strongly antigen-presenting dendritic cells (DC). These monocyte-derived DC (mdDC) were used here to further delineate differentiation pathways in the myeloid lineage. Incubation of mdDC with TNF or soluble CD40L led to enhanced MHC and accessory surface antigen expression with significantly elevated T cell stimulatory activity, indicative of DC maturation. In contrast, after cytokine withdrawal or incubation with M-CSF, mdDC differentiated to macrophages. Cells became adherent, monocyte/macrophage surface markers were upregulated, and MHC and accessory surface proteins were downregulated. Furthermore, the multilaminar MHC class II compartments (MIIC) were lost and the T cell stimulating capacity largely diminished. Thus, mdDC show a high developmental plasticity by retaining their ability to become macrophages or to continue their differentiation towards mature DC.

Antigen Presentation↗

PubMed1997

Identification of individual barley chromosomes based on repetitive sequences: conservative distribution of Afa-family repetitive sequences on the chromosomes of barley and wheat.

The Afa-family repetitive sequences were isolated from barley (Hordeum vulgare, 2n = 14) and cloned as pHvA14. This sequence distinguished each barely chromosome by in situ hybridization. Double color fluorescence in situ hybridization using pHvA14 and 5S rDNA or HvRT-family sequence (subtelomeric sequence of barley) allocated individual barley chromosomes showing a specific pattern of pHvA14 to chromosome 1H to 7H. As the case of the D genome chromosomes of Aegilops squarrosa and common wheat (Triticum aestivum) hybridized by its Afa-family sequences, the signals of pHvA14 in barley chromosomes tended to appear in the distal regions that do not carry many chromosome band markers. In the telomeric regions these signals always placed in more proximal portions than those of HvRT-family. Based on the distribution patterns of Afa-family sequences in the chromosomes of barley and D genome chromosomes of wheat, we discuss a possible mechanism of amplification of the repetitive sequences during the evolution of Triticeae. In addition, we show here that HvRT-family also could be used to distinguish individual barley chromosomes from the patterns of in situ hybridization.

Biological Evolution↗

PubMed1997

HPC-1/syntaxin-1A activity in the enteric nervous system of developing rat gastrointestinal tract.

The HPC-1/syntaxin-1A antigen was originally identified as a neuron-specific membrane protein in the central nervous system. The presence of HPC-1 antigen in the nervous system of the fetal rat gastrointestinal tract was immunohistochemically demonstrated using the antibody against HPC-1 to clarify the role of this protein in the development of the enteric nervous system. Rat gastrointestinal tract from 14-, 16-, 18-, and 20-day fetuses and adults were immunohistochemically examined for HPC-1 antigen by light microscopy. Acetylcholinesterase (AchE) activity was also examined as a comparison. HPC-1 activity was first detected on 18th day of gestation. AchE activity was first detected at the Auerbach's plexus of the esophagus on the 16th day of gestation. The presence of HPC-1 in the developing rat intestine revealed that the HPC-1 antigen may be a good indicator for expressing the maturation of enteric nervous system in the development of the enteric nervous system.

Acetylcholinesterase↗

PubMed1996

Enhancement of neurite-sprouting by suppression of HPC-1/syntaxin 1A activity in cultured vertebrate nerve cells.

HPC-1/syntaxin 1A is a C-terminal anchored neuronal membrane protein, of which all of the N-terminal regions are located on the intracellular side, and it interacts with presynaptic membrane proteins, synaptic vesicle proteins and soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAPs). HPC-1/syntaxin 1A has been proposed to act as a target SNAP receptor (t-SNARE) in the neuron and contributes to the vesicle docking/fusion process during the fast exocytosis at the presynaptic active zone. However, studies using an electron-microscope revealed that HPC-1/syntaxin 1A distributed not only at the presynaptic region but throughout the whole axonal membrane, and the functions of this axonal HPC-1/syntaxin 1A remain completely unknown. To investigate its physiological role, we attempted to inhibit the function of HPC-1/syntaxin 1A in cultured neural cells by following two methods. First, de novo synthesis of HPC-1/syntaxin 1A was inhibited by an application of antisense oligonucleotide in cultured adult rat dorsal root ganglion (DRG) neurons. Second, antibody against HPC-1/syntaxin 1A was applied intra-axonally in the cultured chick retinal ganglion neuron. Both treatments, which were expected to downregulate the function of HPC-1/syntaxin 1A, consistently elicited an enhancement of the axonal sprouting. These results suggest that the axonal HPC-1/syntaxin 1A would physiologically suppress the excess axon-collateral sprouting. Downregulation of HPC-1/syntaxin 1A expression may underlie the control of collateral sprouting and synapse formation during development and memory processes.

Animals↗

PubMed1996

Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway.

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic beta cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of approximately 35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic beta-cells, rat syntaxin 1A or 1B was overexpressed in mouse beta TC3 cells using the transient transfection procedure. Transfection of beta TC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable beta TC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-beta-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected beta TC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing beta TC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.

Amino Acid Sequence↗

PubMed1996

Suppression of superoxide-generating ability during differentiation of monocytes to dendritic cells.

Human peripheral monocytes cultured with GM-CSF and IL-4 differentiated to dendritic cells (DCs) and with GM-CSF alone to macrophages. Superoxide-generating ability in such DCs was found to be suppressed whereas that in macrophages remained constant. To examine the reason for the suppression in DCs, we evaluated by immunoblotting the levels of essential components of the superoxide generating system in the cells during the differentiation. In contrast to the levels of cytosolic 47- and 65-kDa components and Rac-p21, which remained constant throughout cultivation, those of the large and the small subunits of cytochrome b558 were found to decrease quickly by day 2 during cultivation of monocytes with GM-CSF and IL-4. DCs obtained after 7 days of cultivation had lost the large subunit almost completely and most of the small subunit. A cell surface epitope of the cytochrome detected by a monoclonal antibody also decreased during the differentiation. On the other hand, these components, including both subunits of cytochrome b558, were maintained in the cells during differentiation of monocytes to macrophages. These results indicate that the decreased levels of cytochrome b558, especially that of the large subunit, is responsible for the low level of superoxide-generating ability of DCs and that the suppression is caused by IL-4.

Acridines↗

PubMed1996

Hypothermic response of mice to ornithine-containing lipids and to endotoxin.

The hypothermic response of mice to ornithine-containing lipids (Orn-Ls) of the form alpha-N-(3-acyloxyacyl)-ornithine and to endotoxin (Escherichia coli 0111:B4 lipopolysaccharide [LPS]) was studied. After the administration of Orn-L or LPS to C3H/HeSlc mice, body temperature decreases were determined at 30-min intervals by inserting a thermistor into the rectum of each mouse. When Orn-L (750 microg) or LPS (70 microg) was injected into the mice, body temperature decreases of 0.8 and 2.0 degrees C, respectively, occurred 1.8 to 2.0 h later. These body temperature decreases were completely suppressed by the preadministration of indomethacin. When anti-tumor necrosis factor alpha (TNF-alpha) antibody was administered before the administration of Orn-L or LPS, only the body temperature decrease by LPS was suppressed. The body temperature decrease by Orn-L was suppressed by anti-interleukin-1beta (IL-1beta) antibody preadministration. Next, in order to study IL-1beta and TNF-alpha mRNA expression in macrophages, peritoneal macrophages were collected 40 min after the administration of Orn-L or LPS to mice. The expression of IL-1beta mRNA by stimulation with Orn-L was as strong as that by stimulation with LPS, but the expression of TNF-alpha mRNA by stimulation with Orn-L was very weak. Our previous studies of in vitro macrophage activation by Orn-L proved that strong induction of IL-1 and prostaglandin E2 generation by Orn-L occurred (Y. Kawai and K. Akagawa, Infect. Immun. 57:2086-2091, 1989). From these experiments, the weak body temperature decrease in mice caused by Orn-L was found to be mediated by cytokines different from those which mediate the strong body temperature decrease caused by LPS. Namely, it was caused by prostaglandin E2 being mediated by IL-1 but not by TNF-alpha.

Animals↗

PubMed1996

Localization of HPC-1/syntaxin 1 in developing rat cerebellar cortex.

In adult rat cerebellum, HPC-1/syntaxin 1 is detected at high density on the plasma membrane of the non-synaptic region of parallel fibers in addition to the synaptic terminal membranes and the synaptic vesicles (Koh, S., Yamamoto, A., Inoue, A., Inoue, Y., Akagawa, Y., Kawamura, Y., Kawamoto, Y., and Tashiro, Y. (1993). J. Neurocytology 22: 995-1005). To assess the possibility that HPC-1/syntaxin 1 participates in the morphogenesis of the nervous system, we examined changes in the localization of HPC-1/syntaxin 1 during postnatal development of the molecular layer of the rat cerebellum. HPC-1/syntaxin 1 appeared in the granule cells in the outer granule cell layer in 3-days-old rat cerebellum when the formation of synapses and the appearance of a synaptic vesicle protein, synaptophysin, had not yet been observed in the molecular layer. At this stage, the granule cells began to form parallel fibers. Confocal laser microscopy and immuno-electron microscopy showed that HPC-1/syntaxin 1 was localized on the extruding plasma membrane of the granule cells to form parallel fibers. In 8-days-old rats, synapses formed between the parallel fibers and the developing dendrites of Purkinje cells, and the HPC-1 immunoreactivity appeared on the axons of parallel fibers and on the synapses. In 21-days-old rats, the HPC-1/syntaxin is involved in the formation of the molecular layer, especially in the axonal growth of the parallel fibers.

Animals↗

PubMed1995

High potassium promotes differentiation of retinal neurons but does not favor rod differentiation.

Neural retinal cells of newborn rats were cultured under dissociated culture conditions. Differentiation of several types of retinal cells was confirmed by immunohistochemical detection of type-specific neural phenotypes. We used Thy-1.1 antigen as a ganglion cell marker, HPC-1 or GABA as an amacrine cell marker and rhodopsin as a rod cell marker. With a high concentration of potassium (38 mM), expression of the respective neural phenotypes were differentially affected. High K+ increased the number of Thy-1.1 positive cells 6 to 8 fold, and drastically promoted their neurite extension. The same culture conditions, however, reduced considerably the number of rhodopsin positive cells, possibly due to the unique membrane properties of photoreceptors. A high K+ concentration also promoted differentiation of HPC-1 positive and GABA positive cells, but to a lesser extent than the Thy-1.1 positive cells. Several possibilities were examined to understand the effect of a high K+ concentration on retinal neural cells. The total cell number in cultures with a high K+ concentration was approximately half of that in control cultures at day 3 and slightly smaller at day 11, suggesting that high K+ did not have a positive general effect on the proliferation or survival of retinal cells. Naturally occurring neuronal death (apoptosis) is a well-known phenomenon during retinal development. A histochemical method for detecting DNA fragmentation, a step preceding apoptosis, showed that high K+ had no preventive effect. BrdU (bromodeoxyuridine) immunohistochemistry showed that high K+ did not seem to enhance proliferation of neural precursor cells. These results indicate that a high K+ concentration promotes the expression of neuronal phenotypes but is not a favorable condition for rod differentiation. Since a high K+ concentration is considered to induce depolarization of nerve cells, the present results suggest an anterograde influence from surrounding neuronal cells, through chronic depolarization by elevated K+, is essential for the differentiation and maturation of retinal cells.

Animals↗

PubMed1995

Syntaxin 1 (HPC-1) is associated with chromaffin granules.

Syntaxin 1 (HPC-1), a component of the receptor for SNAPs (soluble N-ethylmaleimide-sensitive factor attachment proteins), has been implicated in the docking and fusion of synaptic vesicles with the plasma membrane. It was reported that syntaxin 1 in rat brain and chromaffin cells (PC12) is exclusively located on the plasma membrane (Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259; Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418). By means of biochemical and morphological analyses, we now show that syntaxin 1 is associated with chromaffin granules in the adrenal medulla. This finding raises the possibility that syntaxin 1 in chromaffin cells is a component of vesicle-SNAP receptor as well as one of target-SNAP receptor on the plasma membrane.

Adrenal Medulla↗

PubMed1995

Involvement of HPC-1/syntaxin-1A antigen in transmitter release from PC12h cells.

We examined the effect of antiserum against HPC-1/Syntaxin-1A on the norepinephrine release from digitonin-permeabilized PC12h cells. PC12h cells were permeabilized with digitonin and preincubated with nonimmunized serum or antiserum against HPC-1. The release of norepinephrine was measured in the presence or absence of calcium. The calcium-dependent norepinephrine release was increased in the cells preincubated with anti HPC-1 antiserum. However, with a higher concentration of anti HPC-1 antiserum, the calcium-dependent norepinephrine release was decreased, possibly because of a nonspecific effect. In the case of purified IgG, the same results were obtained. These findings suggested that HPC-1 plays an important role in the exocytosis of transmitter presumably by suppressing the membrane fusion process between the synaptic vesicle and presynaptic membrane.

Analysis of Variance↗