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Biomedical subjects

Y Hata

Publications and source records attributed to Y Hata.

At least 217 records · Page 12Linked to original sources
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

Effects of washing acid injuries to the skin with water: an experimental study using rats.

A skin acid injury model has been constructed using SD rats and 1N HCl. The changes over time, with particular attention to subcutaneous tissue pH, were recorded and included a comparative study of the effect of washing with water at 1, 3 and 10 min after injury, on subcutaneous tissue pH. After inflicting an acid injury, the subcutaneous tissue pH of control animals reached its minimum value at the seventh minute, and had not recovered to the pre-experimental level by the 60th minute. In the group of rats which was washed at 1 min, the pH did not drop below 7.5, it remained virtually unchanged. In the group washed at 3 min, the pH declined slightly, but subsequent to washing, the pH increased gradually. In the group washed at 10 min, the changes in pH were almost the same as those in the untreated group. These results indicated that if washing was carried out before the subcutaneous tissue pH level reached a minimum value, any remaining acid on the skin surface could be washed away, effectively suppressing the subsequent fall in pH.

Animals↗
PubMed1995

Pectoralis major myocutaneous flaps in rats.

A new neurovascular, island, myocutaneous flap, including the pectoralis major, was created in a rat model. This model is useful for the observation of muscle degeneration and skin changes due to ischemia or denervation. Although this model requires a delay procedure, it allows the flap, that can be used as a free myocutaneous flap, to be raised reliably.

Animals↗
PubMed1995

Crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IFO3080 and its conformational changes on ligand binding.

The crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IFO3080 has been determined at 2.0 A resolution by the X-ray method. The enzyme consists of N-terminal catalytic and C-terminal beta-helix domains. On structural comparison between the present unliganded enzyme and structurally- known liganded enzyme, some structural changes were observed around the active site. In the unliganded enzyme, Y216 serves as the fifth ligand for the active site zinc ion. On ligand binding, Y216 may move to form a hydrogen-bond with the carbonyl oxygen of the P1 residue of a ligand peptide. D191 in the flexible loop, Y190 to D196, over the active site cleft forms hydrogen-bonds with the backbone atoms of the P1 and P2 residues of the ligand to close the entrance to the cleft. The water molecule which is the fourth ligand for the zinc ion is replaced by the carbonyl oxygen of the P1 residue. These structural changes around the active site may reflect the substrate-binding mode during the enzymatic reaction.

Bacterial Proteins↗
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

Mechanisms underlying direction selectivity of neurons in the primary visual cortex of the macaque.

1. We studied the effects of blocking intracortical inhibition by microiontophoretic administration of bicuculline methiodide (BMI), a selective antagonist for gamma-aminobutyric acid-A receptors, on direction sensitivity of 103 neurons in the primary visual cortex (VI) of anesthetized and paralyzed monkeys. 2. The direction selectivity index (DSI) of each cell was calculated for the control response and response during the BMI administration at the optimal stimulus orientation to assess the directionality of an individual cell. 3. The averaged direction tuning of visual responses of cells was sharp in layers IVa and IVb, moderate in both interblob and blob regions of layer II/III and layers V and VI, and poor in layers IVc alpha and IVc beta. 4. Iontophoretic administration of BMI uncovered or facilitated responses to stimuli moving in the nonpreferred direction, and reduced DSIs of cells to a varying extent in all the layers except layer VI. Responses to stimuli moving in the preferred direction were also facilitated so that a slight bias of response toward the originally preferred direction remained during BMI administration in most cells. 5. Most of the cells in layers II/III (both blobs and interblobs) and IVb that receive inputs from layers IVc alpha and IVc beta showed a clear reduction of direction selectivity during BMI administration. This result suggests that intracortical inhibition plays an important role in the elaboration of direction selectivity at the second stage of information processing in VI. 6. The direction selectivity of cells in layer VI was most resistant to the effects of BMI, suggesting that it is dependent on excitatory inputs that are already direction selective, even though the sample size of this layer was small. 7. In direction-selective cells outside layer VI, responses to a stimulus moving in the preferred direction were enhanced in a way that was linearly related with those in the nonpreferred direction as the BMI dose was increased. This suggests that various amounts of inhibition interact linearly with directionally biased excitatory inputs to raise the firing threshold to various levels so as to produce various degrees of directionality. 8. These results suggest that, in most of the directionally sensitive cells except for those in layer VI, there are excitatory inputs which are bidirectional but slightly biased to one direction, and that the intracortical inhibition raises a threshold level of responses to excitatory inputs so that the response become direction selective.

Animals↗
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

Serum triglycerides and blood coagulation factors VII and X, and plasminogen activator inhibitor-1.

It has been suggested that impaired fibrinolytic-coagulation system, such as increased concentration of inhibitors to fibrinolysis or activators to coagulations, occasionally may play a role in the development of atherosclerotic vascular disease. In this study, we aimed to elucidate the relationship of serum lipids to fibrinolytic-coagulation system. The subjects studied were 190 outpatients at Kyorin University Hospital, 108 of whom were mostly hypertension, diabetes mellitus and hyperuricemia (Control), 59 of whom were coronary heart disease (CHD), 25 of whom were cerebrovascular disease (CVD). Blood samples were measured the levels of blood coagulation factors VII (F-VII) and X (F-X), and plasminogen activator inhibitor-1 (PAI-1) in these subjects, together with the concentrations of serum lipids. The serum levels of F-X was significantly higher in CHD subjects than in controls (111 +/- 19% vs 101 +/- 22%, p < 0.05). However, there was a no significant difference of F-VII among three groups. And we found that the levels of serum lipids, especially serum triglycerides showed a significant positive correlation between the concentrations of F-VII (r = 0.343, p < 0.01) and F-X (r = 0.513, p < 0.01), and PAI-1 (r = 0.528, p < 0.001) in CHD and CVD subjects. For this reason, 156 bank employee subjects were also admitted to this study (Bank employees). In bank employee subjects, the serum levels of triglycerides also showed a significant positive correlation with the levels of F-VII (r = 0.321, p < 0.001), F-X (r = 0.254, p < 0.001) and PAI-1 (r = 0.420, p < 0.001). These data suggest that serum lipids, particularly triglycerides have a close relationship with thrombogenesis as evidenced by activated F-VII and F-X in the extrinsic coagulation system and also by elevated PAI-1 activities in fibrinolysis. Therefore, when we try to prevent the patients from CHD or treat them, we ought pay attentions not only to serum cholesterol or LDL-cholesterol for their atherogenic actions, but also to triglycerides because of their close correlation with extrinsic coagulation system and anti-fibrinolytic activities. The reduction of fibrinolytic capacity due to increased plasma levels of F-VII, X and PAI-1 may have importance in atherosclerotic vascular disease, particularly in patients with hypertriglyceridemia.

Cerebrovascular Disorders↗
PubMed1995

Hypertriglyceridemia and fatty liver: clinical diagnosis of fatty liver and lipoprotein profiles in hypertriglyceridemic patients with fatty liver.

Fatty liver has prevailed by 14% in the healthy population of this country. The factors contributing genesis of fatty liver were gender (male), obesity, high alcohol consumption, glucose intolerance and hypertriglyceridemia. And hypertriglyceridemia seems to be the common underlying factor to all other causes. The mechanism for accumulation of triglycerides in the liver can be explained at least by increased HTGL activities and elevated apo A-II levels, a postulated co-factor of HTGL. An hypertriglyceridemic patients with fatty liver had the insulin resistance.

Adult↗
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↗