Biomedical subjects

K Akagawa

Publications and source records attributed to K Akagawa.

At least 19 recordsLinked to original sources

PubMed1999

HPC-1/syntaxin 1A suppresses exocytosis of PC12 cells.

The membrane protein syntaxin (originally named HPC-1) is involved in vesicle trafficking and required for neurotransmitter release at nerve terminals. The presence of syntaxin on target membranes is hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins. To elucidate the function of syntaxin 1A in exocytosis, HPC-1/syntaxin 1A-reduced PC12h cells (PC12h/Deltasyx) that were stably transfected with a plasmid for antisense syntaxin 1A expression were constructed. Depolarizing stimulation of PC12h/Deltasyx enhanced dopamine release, compared with PC12h. There was a strong inverse correlation between syntaxin 1A protein expression and enhancement of dopamine release. Reduction of syntaxin 1A had no effect on increase of the cytoplasmic free Ca2+ concentration by depolarized stimulation. Moreover, PC12h/Deltasyx clones similarly enhanced of exocytosis by native secretagogues. These results indicate that syntaxin 1A has more than one function in exocytosis.

Animals↗

PubMed1999

A typical bacterial ornithine-containing lipid Nalpha-(D)-[3-(hexadecanoyloxy)hexadecanoyl]-ornithine is a strong stimulant for macrophages and a useful adjuvant.

Nalpha-[3-(Hexadecanoyloxy)hexadecanoyl]-ornithine is a typical bacterial ornithine-containing lipid (OL). The configuration of the 3-hydroxy fatty acids in the OL was proved to be D by using HPLC with chiral column. For this analysis, Nalpha-(D or L)-[3-(hexadecanoyloxy)hexadecanoyl]-L-ornithine were synthesized and used as standards. The typical bacterial OL, as well as the synthesized one, exhibited strong interleukin-1- and prostaglandin E2-inducing activities, and further, it induced the production of high IgG anti-tetanus toxoid antibodies in mice. The typical OL is expected to be utilized as a nontoxic, potent adjuvant.

Adjuvants, Immunologic↗

PubMed1998

Differential expression of rat brain synaptic proteins in development and aging.

We have previously reported the differential involvement of synaptic proteins in Alzheimer's disease (AD). As AD is an aging-associated disease, in the present study we examined the developmental and aging-related changes in synaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), syntaxin 1/HPC-1 and drebrin in the rat brain. Immunoblot analyses of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that the protein level of synaptophysin and synaptobrevin increased after birth, being highest at 24 weeks, and then decreased with aging. Synaptotagmin was detected at E19, with levels increasing after birth to 96 weeks. SNAP-25 levels were highest at 4 weeks, and then decreased with aging. Syntaxin 1/HPC-1 levels were high at E19 and 1 week, decreasing rapidly from 2 weeks onwards, and drebrin levels were highest at E19 and 1 week, and decreased during aging. The present results suggest that the expression of each synaptic protein is differentially regulated in development and aging.

Aging↗

PubMed1998

Important roles of the C-terminal portion of HPC-1/syntaxin 1A in membrane anchoring and intracellular localization.

HPC-1/syntaxin 1A (HPC-1), which plays an important role in vesicular transport to the plasma membrane, possesses a hydrophobic sequence at its C terminus. When expressed from cDNA in COS cells, wild-type HPC-1 was localized in the Golgi complex and the plasma membrane. Truncation of the hydrophobic domain resulted in the cytoplasmic localization of the mutant, thus indicating that the domain indeed functions as a membrane anchor. A fusion protein with the C-terminal glycosylation sites was glycosylated in transfected cells, providing evidence that HPC-1 has a transmembrane structure, and that the protein is first inserted into the endoplasmic reticulum and then transported to the plasma membrane. A chimeric protein consisting of Escherichia coli maltose-binding protein with the last 24 amino acids of HPC-1 was inserted into the endoplasmic reticulum in a transmembrane topology and localized along the exocytic pathway of transfected cells similar to HPC-1. These results indicate that the portion is important for intracellular localization of HPC-1.

Amino Acid Sequence↗

PubMed1998

Hemizygous deletion of the HPC-1/syntaxin 1A gene (STX1A) in patients with Williams syndrome.

HPC-1/syntaxin 1A is a membrane protein that plays an important role in exocytosis of neurotransmitters from neuronal cells. We previously mapped the human HPC-1/syntaxin 1A gene (STX1A) to chromosome 7q11.2, which is within the Williams syndrome (WS) region. Here, we performed FISH analysis on 46 patients with WS to examine the relationship between STX1A and WS. Our results showed a hemizygous deletion of the HPC-1/syntaxin 1A gene in each patient, suggesting that the neurological symptoms of WS may be related to the hemizygous deletion of STX1A.

Adolescent↗

PubMed1997

Efficient virus transmission from dendritic cells to CD4+ T cells in response to antigen depends on close contact through adhesion molecules.

Monocyte-derived cultured dendritic cells (DCs) are potent antigen-presenting cells (APCs) and are susceptible to HIV-1Lai infection. Compared to the low level of virus production by HIV-1-infected DCs alone, a level of virus two to three orders of magnitude higher was produced by cocultivation of HIV-1-infected DCs with autologous resting CD4+ T cells in the presence of a nominal antigen. In this coculture system, direct contact of HIV-1-infected DCs with T cells was crucial for efficient virus transmission and subsequent virus production. Blocking of the LFA-1/ICAM-1 or LFA-3/CD2 interaction between these cells substantially reduced virus production, without influence or IL-2 production by activated T cells. In contrast, cell-cell transmission of HIV between non-APCs and activated T cells was not blocked by an antibody against LFA-3. Since a low level of virus production by HIV-infected DCs was upregulated by cross-linking of CD40, it was suggested that not only focal adhesion, but also mutual activation of HIV-infected DCs and T cells through adhesion molecules, may potentiate virus transmission and production and that such activation signals to HIV may be distinct from signals responsible for IL-2 production in activated T cells.

Antigen Presentation↗

PubMed1997

Enhancement of synaptic transmission by HPC-1 antibody in the cultured hippocampal neuron.

To clarify the function of HPC-1/syntaxin 1A in the mammalian central synapse, the effects of intracellularly applied antibody on the synaptic transmission were examined at the autapse of the cultured rat hippocampal neuron. Intracellularly applied antibody against HPC-1/syntaxin 1A (IgG, 0.3 mg ml(-1)) during whole-cell recording enhanced the autaptic excitatory postsynaptic current (EPSC). Pre-immune IgG (0.3 mg ml(-1)) showed no effect. The amplitude-distribution of an asynchronous EPSC was not affected by administration of this antibody, indicating that the increase in the amplitude of the evoked EPSC was attributable to an increase in transmitter release from the presynaptic terminal HPC-1/syntaxin 1A could be involved in suppressing as well as facilitating process of the exocytosis at the mammalian central synapse.

Animals↗

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↗