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Biomedical subjects

Y Hata

Publications and source records attributed to Y Hata.

At least 19 recordsLinked to original sources
PubMed1995

Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins.

The unc-13 gene in Caenorhabditis elegans is essential for normal presynaptic function and encodes a large protein with C1- and C2-domains. In protein kinase C and synaptotagmin, C1- and/or C2-domains are regulatory domains for Ca2+, phospholipids, and diacylglycerol, suggesting a role for unc-13 in regulating neurotransmitter release. To determine if a similar protein is a component of the presynaptic machinery for neurotransmitter release in vertebrates, we studied unc-13 homologues in rat. Molecular cloning revealed that three homologues of unc-13 called Munc13-1, -13-2, and -13-3 are expressed in rat brain. Munc13s are large, brain-specific proteins with divergent N termini but conserved C termini containing C1- and C2-domains. Specific antibodies demonstrated that Munc13-1 is a peripheral membrane protein that is enriched in synaptosomes and localized to plasma membranes but absent from synaptic vesicles. Our data suggest that the function of unc-13 in C. elegans is conserved in mammals and that Munc13s act as plasma membrane proteins in nerve terminals. The presence of C1- and C2-domains in these proteins and the phenotype of the C. elegans mutants raise the possibility that Munc13s may have an essential signaling role during neurotransmitter release.

Amino Acid Sequence↗
PubMed1995

High affinity binding of alpha-latrotoxin to recombinant neurexin I alpha.

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity alpha-latrotoxin binding proteins were isolated and demonstrated to contain neurexin I alpha as a major component. Neurexin I alpha is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin I alpha binds alpha-latrotoxin directly with high affinity (Kd approximately 4 nM). Binding of alpha-latrotoxin to recombinant neurexin I alpha is dependent on Ca2+ (EC50 approximately 30 microM). Our data suggest that neurexin I alpha is a Ca(2+)-dependent high affinity receptor for alpha-latrotoxin.

Alternative Splicing↗
PubMed1995

A novel ubiquitous form of Munc-18 interacts with multiple syntaxins. Use of the yeast two-hybrid system to study interactions between proteins involved in membrane traffic.

Munc-18-1 is a 67-kDa neuronal protein that binds tightly to syntaxin 1 and functions in synaptic vesicle exocytosis (Hata, Y., Slaughter, C.A., and Südhof, T.C. (1993a) Nature 366, 347-351). We have now characterized a new Munc-18 isoform, Munc-18-2, that exhibits 63% amino acid sequence identity with Munc-18-1. Munc-18-2 is expressed in most tissues, whereas Munc-18-1 is primarily expressed in brain. Using recombinant Munc-18-1 and Munc-18-2 produced in COS cells, we show that both forms of Munc-18 bind tightly to syntaxins 1A, 2, and 3 but not to syntaxin 4. In an independent approach to study the binding specificities of Munc-18-1 and Munc-18-2, we used the yeast two-hybrid system. This assay system depends on protein-protein interactions in the cell nucleus. We validated its utility for studying membrane trafficking proteins by testing well characterized interactions between cytosolic proteins that are known to be physiologically important in exocytosis. Strong interactions, such as the binding of syntaxins 1-4 with SNAP-25, were effectively detected by the yeast two-hybrid assay, but weak binding, such as the binding of syntaxins to synaptotagmin or of synaptotagmin to neurexins, was not. Studies on full-length and truncated forms of Munc-18s by the yeast two-hybrid system confirmed their interactions with syntaxins. Both the N and the C terminus of Munc-18 were essential for binding. Munc-18-1 and Munc-18-2 bind only to syntaxins 1A, 2, and 3 but not 4 and 5 by yeast-two hybrid system assays. Our studies demonstrate that neural and non-neural tissues have distinct forms of Munc-18, which may function in different types of exocytosis. The lack of specificity of the interactions between syntaxins and Munc-18s indicates that specificity of membrane trafficking reactions is not dependent on this interaction.

Amino Acid Sequence↗
PubMed1995

Neuroligin 1: a splice site-specific ligand for beta-neurexins.

Neurexins are neuronal cell surface proteins with hundreds of isoforms generated by alternative splicing. Here we describe neuroligin 1, a neuronal cell surface protein that is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds to beta-neurexins only if they lack an insert in the alternatively spliced sequence of the G domain, but not if they contain an insert. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. In situ hybridization reveals that alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. These findings support a model whereby alternative splicing of neurexins creates a family of cell surface receptors that confers interactive specificity onto their resident neurons.

Alternative Splicing↗
PubMed1995

Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis.

To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (20% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P < 0.01) and in vitro angiogenesis (2.4 times, P < 0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner.

Animals↗
PubMed1995

Early afterdepolarization abolished by potassium channel opener in a patient with idiopathic long QT syndrome.

We describe a 17-year-old boy with idiopathic long QT syndrome and repeated syncopal episodes. Early afterdepolarization (EAD) in the monophasic action potential (MAP) was demonstrated in the posterior septum of the left ventricle. Injection of the potassium channel opener nicorandil decreased EAD and shortened MAP duration. The syncopal episodes due to ventricular fibrillation disappeared after administration of the potassium channel opener.

Action Potentials↗
PubMed1995

Vascular endothelial growth factor plays a role in hyperpermeability of diabetic retinal vessels.

In rat diabetic retinas, we immunohistochemically looked for vascular endothelial growth factor (VEGF) which is also known as vascular permeability factor (VPF). In nondiabetic retinas, VEGF immunoreactivity was weak and restricted to the nerve fiber and ganglion cell layers. On the other hand, in diabetic retinas, VEGF immunoreactivity was markedly increased and was observed in all layers of the retina, especially in the perivascular area. Hyperpermeability of these vessels was confirmed by immunohistochemically detecting extravasation of albumin. These findings indicate that vascular endothelial growth factor plays an important role in blood-retinal barrier breakdown in diabetic retinopathy.

Actins↗
PubMed1995

[Evaluation of the furosemide test using the air caloric stimulator].

Both the furosemide test, and the glycerol test have been reported as being effective for detection of endolymphatic hydrops. In the furosemide test, the caloric test is performed first, and then repeated 40 minutes after i.v. injection of furosemide, 20 mg. The maximum velocities of each caloric nystagmus are compared, so the caloric stimulus conditions must be constant. In the original method, the caloric test is performed by irrigation with 50 ml of water at 30 degrees C or 44 degrees C for 20 seconds. Since it is difficult to accurately maintain the water temperature at a constant level, however, we use the air caloric stimulator NCA-105 (ICS) for the furosemide test. By using this stimulator, we can always perform the caloric test under the same conditions. The conditions of air irrigation in 28 normal subjects were set at 37 +/- 11 degrees C, 61/min, and 60 seconds. These conditions were confirmed in other normal subjects to fairly well correspond to water irrigation (30 degrees C or 44 degrees C, 50 ml, 20 seconds). Next we reassessed the furosemide test by using the air caloric stimulator in normal subjects. As a result, positive effects were observed in 7.4% of the normal subjects. This finding was similar to that reported in the literature (Futaki et al., 1971). The air caloric test is considered to be a more useful examination than the water caloric test because it is less unpleasant for the subject and easily provides the same conditions before and after furosemide administration.

Adolescent↗
PubMed1995

Photocoagulated human retinal pigment epithelial cells produce an inhibitor of vascular endothelial cell proliferation.

PURPOSE: To determine if conditioned culture medium of photocoagulated human retinal pigment epithelial (RPE) cells contains inhibitors for the proliferation of bovine aortic endothelial cells (BAEC) and bovine retinal endothelial cells (BREC) and to characterize the nature of these inhibitory factors. METHODS: Retinal pigment epithelial cells grown to confluence were photocoagulated (0.1 second, 50 microns, 350 mW) in serum-free medium. After 48 hours, the conditioned medium (PC-CM) was removed, and the effects of non-acid-treated and transiently acid-treated samples were determined on [3H]-thymidine uptake by BAEC and BREC. PC-CM was also subjected to size exclusion high-performance liquid chromatography (HPLC). Fractions were analyzed for the effects on the growth of the bovine endothelial cells before and after transient acid treatment. RESULTS: The addition of non-acid-treated PC-CM (32% vol/vol) inhibited BREC [3H]-thymidine uptake to 18.5% of the control value. With HPLC, the inhibitory activity was recovered mainly in a fraction whose apparent molecular size was 25 kd. After transient acid treatment of the fractions, there also appeared a 100-kd inhibitor. Inhibitory effects were neutralized by pretreatment of the fractions with antiserum against transforming growth factor (TGF)-beta 2. CONCLUSIONS: Photocoagulated RPE cells secrete inhibitors of BAEC and BREC proliferation. Molecular size and immunologic properties of these inhibitors correspond to those of TGF-beta 2.

Animals↗
PubMed1994

Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.

Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP-25 and syntaxin, and SNAP-25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate-resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha-helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled-coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin-syntaxin-SNAP-25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP-25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.

Animals↗
PubMed1994

Control of thalamocortical afferent rearrangement by postsynaptic activity in developing visual cortex.

The formation of specific connections in the developing central nervous system is thought to result from mechanisms that increase the strengths of synapses at which pre- and postsynaptic activity are correlated and decrease it otherwise. In the visual cortex, initially widespread inputs normally sort out into eye-specific patches during early life. If only one eye can see during this period, its patches are much larger than normal, and patches from the occluded eye become much smaller. Anatomical experiments here show that closed-eye inputs expand within a region of cortex that is silenced, establishing that inhibition of common target cells gives less active inputs a competitive advantage.

Animals↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of an endoglucanase from Aspergillus aculeatus.

Most fungal cellulases are found in multiple forms varying in size and substrate specificity. Aspergillus aculeatus is known to produce nine cellulolytic enzymes including an endoglucanase (FI CM-cellulase, M(r) = 24,002) as the major component. Single crystals of FI CM-cellulase from Aspergillus aculeatus have been prepared by sitting-drop vapour diffusion using ammonium sulphate as a precipitant. The cellulase crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 52.79(2) A, b = 106.40(4) A and c = 33.15(1) A. The crystals contain one enzyme molecule per asymmetric unit. They diffract to at least 2.0 A resolution and are very stable against X-ray irradiation.

Ammonium Sulfate↗
PubMed1994

Structural characteristics for biological activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli: X-ray crystallography of weakly toxic and nontoxic analogs.

Heat-stable enterotoxin (ST) produced by a pathogenic strain of Escherichia coli exerts its function by binding to a membrane-bound guanylyl cyclase on intestinal epithelial cell membranes, which in turn catalyzes the production of cyclic GMP as a second messenger in the cells. To elucidate the structural requirements for the biological activities of ST, we synthesized [Mpr5,Gly13]STp(5-17) and [Mpr5,Leu13]STp(5-17), which are weakly toxic and nontoxic analogs of STp, in which the toxic domain consists of the sequence from Cys at position 5 to Cys at position 17. In these analogs, Cys at position 5 is replaced by Mpr (beta-mercaptopropionic acid) and Ala at position 13 by Gly and Leu, respectively. We examined these analogs by X-ray diffraction analysis using direct methods and refined the structures to crystallographic R factors of 7.3% and 6.6% using 5492 and 5122 data, respectively, observed > 3 sigma (Fo) with a resolution of 0.89 A. These peptides have a right-handed spiral structure consisting of three structural segments: an N-terminal 3(10) helix, a central type I beta-turn, and a C-terminal type II beta-turn. These structures show minor differences from that of [Mpr5]STp(5-17), the fully toxic analog of heat-stable enterotoxin [Ozaki et al. (1991) J. Biol. Chem. 266, 5934-5941], suggesting that the decrease and loss of the biological activities of [Mpr5,Gly13]STp(5-17) and [Mpr5,Leu13]STp(5-17), respectively, are not caused by structural changes but are associated with the direct interaction of Ala13 with the receptor protein. Careful comparison of these structures in crystalline states revealed that ST has the following structural characteristics: (i) inherent flexibility at the junctions of the three segments and in the central segment, which includes the putative receptor-binding residues, Ala13, (ii) a specific hydrophobic character around the central segment, and (iii) an unexpected C-terminal folding similar to those of functionally unrelated peptides that are known to be ionophores.

Amino Acid Sequence↗
PubMed1994

Conserved domain structure of beta-neurexins. Unusual cleaved signal sequences in receptor-like neuronal cell-surface proteins.

Neurexins, a family of neuronal cell-surface proteins, consist of the longer alpha-neurexins (I alpha, II alpha, and III alpha) and the shorter beta-neurexins (I beta and II beta) with identical C termini but distinct N termini. alpha-Neurexins have the structure of cell surface receptors, but the membrane topology and conservation of beta-neurexins is unknown. We have now characterized cDNA clones encoding bovine neurexins I beta and III beta, thereby demonstrating the presence of a beta-form for neurexin III and the evolutionary conservation of beta-neurexins in mammals. Similar to alpha-neurexins, beta-neurexins were found to be highly O-glycosylated after expression by transfection in COS cells, suggesting that alpha- and beta-neurexins utilize the same O-glycosylation cassette and have similar transmembrane orientations. To determine if beta-neurexins contain a cleaved or uncleaved signal sequence for membrane translocation, beta-neurexin-IgG fusion proteins were expressed in COS cells, and their N termini were directly sequenced. This revealed that the N terminus of all three beta-neurexins contains an unusual cleaved signal sequence. Together our data show that all known neurexin genes generate alpha and beta forms with similar transmembrane organizations and receptor-like structures. Due to the presence of a long atypical cleaved signal peptide, beta-neurexins contain only a short unique sequence before splicing into the alpha-neurexin sequence. Thus, beta-neurexins are essentially N terminally truncated alpha-neurexins.

Amino Acid Sequence↗
PubMed1994

Role of differentiation induction in action of purine antimetabolites.

In cancer cells, particularly in leukaemic cells, guanylate biosynthesis is up-regulated as shown by the increased activities of IMP dehydrogenase, the rate-limiting enzyme of de novo GTP biosynthesis, and of the salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). In enzyme pattern-targeted chemotherapy, tiazofurin inhibits IMP dehydrogenase activity in cancer cells and allopurinol-induced high serum hypoxanthine levels inhibit HGPRT activity. A triad of responses was observed in the blast cells of patients treated with tiazofurin infusions: chemotherapy, induced differentiation, and down-regulation of c-Ki-ras and c-myc oncogenes. Tiazofurin was synergistic in cytotoxicity and in causing differentiation with ribavirin, retinoic acid, and gemcitabine [corrected]. Induced differentiation plays an important role in the overall impact of antipurine agents.

Animals↗
PubMed1994

Growth regulation of rabbit gastric epithelial cells and protooncogene expression.

We recently developed a primary culture system for gastric epithelial cells from adult rabbits that allows the investigation of growth regulation at the cellular level. In this study, we demonstrated that epidermal growth factor (EGF), insulin, and dibutyryl adenosine 3',5'-cyclic monophosphate (dBcAMP) all stimulated cell proliferation. Insulin and dBcAMP potentiated the stimulation of cell proliferation by EGF, while transforming growth factor-beta 1 (TGF-beta 1) inhibited it. Expression of c-fos and c-myc was induced in response to the stimulation by these growth regulators, but the degree of expression did not necessarily correlate with the effects of these agents on cell proliferation. In conclusion, EGF, insulin, and dBcAMP were positive growth regulators, while TGF-beta 1 was a negative regulator in gastric epithelial cells. These growth modulators may exert their effects by distinct pathways from a standpoint of the expression of c-fos and c-myc.

Animals↗
PubMed1994

Pseudomyxoma peritonei due to adenocarcinoma of the lung: case report.

A rare case of pseudomyxoma peritonei whose primary site was presumed to be the lung is reported. A 76-year-old woman was admitted to Hospital presenting with progressive abdominal distention. She had been admitted twice, 2 and 1 year previously for the evaluation of high plasma carcinoembryonic antigen (CEA) level, of 11.6 ng/ml. Chest computed tomography (CT) scan and chest X-ray film on the third admission revealed a nodular lesion in the left lower lung field, and transbronchial lung biopsy (TBLB) revealed mucus-producing tall columnar epithelial carcinoma. Paracentesis revealed gelatinous ascitic fluid. At laparotomy, appendix and ovary were normal, and there were many small cystic tumors on the peritoneal surface and omentum. The patient died 2 years later, after repeated episodes of dynamic ileus. The lung and abdominal tumors gradually increased in size during the 2-year period, but she developed no respiratory symptoms. Based on both the clinical and pathophysiological findings, the final diagnosis made was pseudomyxoma peritonei whose origin was a lung adenocarcinoma.

Adenocarcinoma↗