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S Mochida

Publications and source records attributed to S Mochida.

At least 19 recordsLinked to original sources
PubMed2001

SNAP-29: a general SNARE protein that inhibits SNARE disassembly and is implicated in synaptic transmission.

Using the yeast two-hybrid system with syntaxin-1A as bait, we isolated soluble NSF attachment protein (SNAP)-29 from a human brain cDNA library. Synaptosomal fractionation and immunocytochemical staining of hippocampal neurons in culture showed that SNAP-29 is present at synapses and is predominantly associated with synaptic vesicles. The interaction of SNAP-29 with syntaxin-1 was further confirmed with immunoprecipitation analysis. Binding competition studies with SNAP-29 demonstrated that it could compete with alpha-SNAP for binding to synaptic SNAP receptors (SNAREs) and consequently inhibit disassembly of the SNARE complex. Introduction of SNAP-29 into presynaptic superior cervical ganglion neurons in culture significantly inhibited synaptic transmission in an activity-dependent manner. Although SNAP-29 has been suggested to be a general SNARE component in membrane trafficking, our findings suggest that it may function as a regulator of SNARE complex disassembly and modulate the process of postfusion recycling of the SNARE components.

Animals↗
PubMed2001

Apoptosis in dissociation between DNA synthesis and cellular functions of activated hepatic stellate cells--a study with carbon tetrachloride-induced rat liver injury.

It is widely believed that DNA synthesis and expressions of smooth muscle alpha actin and TGF-beta are all together increased in activated hepatic stellate cells both in vitro and in vivo. Our previous reports disclosed that these increases did not always coexist under experimental conditions. Liver necrosis was induced in rats by oral administration of carbon tetrachloride. Hepatic stellate cells were isolated from these rats 2 days later. When these cells were cultured on plastic dishes for 3 days, they showed marked DNA synthesis and smooth muscle alpha actin and TGF-beta mRNA expressions assessed by (3)H-thymidine incorporation and Northern blotting, respectively. In the cells further cultured for 7 days, the DNA synthesis was decreased, whereas both smooth muscle alpha actin and TGF-beta mRNA expressions were increased, compared to the cells cultured for 3 days. The cells cultured for 10 days showed apoptotic nuclei positive for nick-end labeling, and DNA extracted from the cells revealed laddering patterns on agarose gels by electrophoresis. Apoptotic nuclei were also immunohistochemically found in stellate cells in the liver of rats 4 days after the intoxication. We conclude that apoptosis developed in activated hepatic stellate cells both in vitro and in vivo, and this may contribute to the discrepancy between DNA synthesis and cellular functions of the cells.

Actins↗
PubMed2001

VEGF can act as vascular permeability factor in the hepatic sinusoids through upregulation of porosity of endothelial cells.

VEGF is shown to be a vascular permeability factor (VPF) as well as a growth stimulatory factor on endothelial cells. In the hepatic sinusoids, endothelial cells express flt-1 and KDR/flk-1, receptors for VEGF. These cells, in primary culture, proliferate in response to VEGF stimulation. However, the role of VEGF as VPF in the hepatic sinusoids is to be elucidated. The effect of VEGF on the porosity of sinusoidal endothelial cells was studied. Sinusoidal endothelial cells were isolated from rats and cultured in DMEM containing 10% FCS on plastic dishes coated with type I collagen for 16 and 48 h for morphological examination and cell-number measurement, respectively. When the cells were cultured without VEGF addition, their number was decreased at 48 h compared to that at 16 h. However, the number was unchanged in the cells cultured with VEGF at 10 ng/mL and increased with addition of VEGF at 100 ng/mL. Scanning electron microscopic examination revealed that sieve-plate appearance of the cells was impaired in culture with no VEGF addition, but the appearance was maintained in culture with VEGF at 10 ng/mL or more. The cells cultured with VEGF at 100 ng/mL showed significantly increased number and size of pores compared to the cells cultured with VEGF at 10 ng/mL, suggesting that sinusoidal endothelial cells proliferating in response to VEGF may increase their porosity. It is concluded that VEGF can act as VPF in the hepatic sinusoids through regulation of endothelial cell porosity.

Animals↗
PubMed2001

TT virus infection in cases of fulminant hepatic failure-evaluation by clonality based on amino acid sequence of hypervariable regions.

The significance of TT virus (TTV) infection in the pathogenesis of acute liver disease is uncertain. Serum TTV-DNA was determined by polymarase-chain reaction (PCR) methods using both hemi-nested (NG059/NG063 and NG061/NG063) and single-step (T801/T935) primers in four patients with fulminant hepatic failure and one patient with late onset hepatic failure in whom hepatitis A virus (HAV) and hepatitis B virus (HBV) markers were negative. Of these five patients, the TTV-DNA was positive in two patients with fulminant hepatic failure by both PCRs before receiving blood transfusion and/or blood-products infusion. Therapies including plasma exchange with blood hemodiafiltration was performed in both patients, and one survived. The non-survivor was an 18-year-old woman with TTV genotype 2 infection. In this patient, the TTV-DNA by PCRs with both primers was positive transiently when serum ALT levels were elevated. The survivor was a 78-year-old woman with infections of TTV genotype 1b and hepatitis C virus (HCV), in whom serum ALT levels returned to normal at 3 weeks after the start of the therapies, but fluctuated after 10 weeks. TTV-DNA measured by hemi-nested primers and HCV-RNA were negative when serum ALT levels decreased, but became positive later. Semi-quantitative PCR using single-step primers revealed that serum TTV-DNA levels changed in correlation with both serum ALT and HCV-RNA levels. Amino acid sequences of hypervariable regions were identical in six out of nine clones isolated from the sera before the therapies and sequence divergence was minimal even in the other three clones, suggesting that TTV proliferated with clonality in the process of fulminant hepatic failure. We conclude that clonally proliferating TTV may contribute to fulminant hepatic failure as a solitary infectious agent or a co-infectious agent with HCV.

Journal Article↗
PubMed2000

Increased expression of osteopontin in activated Kupffer cells and hepatic macrophages during macrophage migration in Propionibacterium acnes-treated rat liver.

Osteopontin is an extracellular matrix component that can act as a chemokine to induce macrophage migration. The significance of osteopontin in macrophage infiltration into the liver was examined in rats given heat-killed Propionibacterium acnes. In normal rats, osteopontin mRNA expression in the liver was minimal, determined by quantitative-competitive reverse transcription-polymerase chain reaction (RT-PCR) assay. Northern blot analysis revealed that osteopontin mRNA was not expressed in Kupffer cells isolated from normal rats. When rats received heat-killed P. acnes intravenously, marked macrophage accumulation, forming granulomas, developed in the liver later than 3 days after the injection and its extent became maximal between 5 and 7 days. In these rats, osteopontin mRNA expression was increased in the liver later than 1 day (with its peak at 3 days after the injection), and the mRNA expression was increased markedly in Kupffer cells and hepatic macrophages isolated at 7 days. The mRNA expression of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), chemokines for monocytes and macrophages, was also increased in the liver of P. acnes-treated rats, with peak expression at 3 days. We conclude that osteopontin derived from Kupffer cells and hepatic macrophages may contribute to the infiltration of monocytes and macrophages into the liver cooperatively with the actions of MCP-1 and MIP-1alpha in P. acnes-treated rats.

Animals↗
PubMed2000

Protein-protein interactions in neurotransmitter release.

The arrival of a nerve impulse at a nerve terminal leads to the opening of voltage-gated Ca(2+) channels and a rapid influx of Ca(2+). The increase in Ca(2+) concentration at the active zone from the basal level of 100-200 mM triggers the fusion of docked synaptic vesicles, resulting in neurotransmitter release. A large number of proteins have been identified at nerve terminals and a cascade of protein-protein interactions has been suggested to be involved in the cycling of synaptic vesicle states. Functional studies in last half decade on synaptic-terminal proteins, including Ca(2+) channels, have revealed that the SNARE core complex, consisting of synaptobrevin VAMP, a synaptic vesicle-associated protein, syntaxin and SNAP-25, synaptic membrane-associated proteins, acts as the membrane fusion machinery and that proteins interacting with the SNARE complex play essential roles in synaptic vesicle exocytosis by regulating assembly and disassembly of the SNARE complex.

Animals↗
PubMed2000

Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly.

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.

Amino Acid Sequence↗
PubMed2000

Biological activities of novel lipid mediator sphingosine 1-phosphate in rat hepatic stellate cells.

Sphingosine 1-phosphate (S-1-P), a lipid mediator shown to be a ligand for aortic G protein-coupled receptor [corrected] (AGRs), endothelial differentiation gene (EDG)1, EDG3, and AGR16/EDG5, is stored in platelets and released on their activation. Platelet consumption occurs in acute liver injury. Hepatic stellate cells (HSCs) play an important role in wound healing. Effects of S-1-P on HSCs were investigated. S-1-P enhanced proliferation of culture-activated HSCs. The mitogenic effect was pertussis toxin sensitive, mitogen-activated protein kinase dependent, and more prominent at lower cell density. S-1-P increased contraction of collagen lattices containing HSCs, irrespective of activation state, in a C3 exotoxin-sensitive manner. mRNAs of EDG1 and AGR16, but not of EDG3, were detected in HSCs. In HSC activation, EDG1 mRNA levels were downregulated, whereas AGR16 mRNA levels were unchanged. Considering that HSCs are capable of production of extracellular matrices and modulation of blood flow in sinusoids, our results suggest that S-1-P may play a role in wound healing process in the liver.

Actins↗
PubMed1999

Inhibition of hepatic stellate cell contraction during activation in vitro by vascular endothelial growth factor in association with upregulation of FLT tyrosine kinase receptor family, FLT-1.

Activated hepatic stellate cells produce vascular endothelial growth factor (VEGF). VEGF has been shown to act on mesenchymal cells as well. If hepatic stellate cells can express FLT tyrosine receptor family, flt-1 and KDR/flk-1, their function might be regulated by VEGF in an autocrine manner. This hypothesis was tested using hepatic stellate cells isolated from normal rats. Northern blot analysis and immunocytochemical study revealed that hepatic stellate cells cultured for 3 days on plastic dishes expressed both flt-1 and KDR/flk-1. When the culture was prolonged to 10 days, the flt-1 mRNA expression was increased, whereas both KDR/flk-1 mRNA and protein expressions diminished. DNA and collagen syntheses were minimal in the cells cultured for 3 days, but marked in those cultured for 10 days. Addition of recombinant human VEGF to the culture medium did not change both syntheses but attenuated an increase of smooth muscle alpha-actin expression in the cells during culture on plastic dishes and also contraction of collagen gels on which the cells were cultured. We conclude that VEGF may inhibit contraction of hepatic stellate cells appearing during activation by culture, probably through attenuation of smooth muscle alpha-actin expression via upregulated VEGF receptor, flt-1.

Animals↗
PubMed1999

Expression of osteopontin in Kupffer cells and hepatic macrophages and Stellate cells in rat liver after carbon tetrachloride intoxication: a possible factor for macrophage migration into hepatic necrotic areas.

Activated Kupffer cells and macrophages accumulate in necrotic areas in the liver. Osteopontin, an extracellular matrix with RGD sequence, has been shown to act as a chemokine that can induce monocyte migration. The possibility that osteopontin can play a role in infiltration of both cells into hepatic necrotic areas was investigated in rats. Northern blot analysis revealed that osteopontin mRNA expression was minimal in Kupffer cells and hepatocytes immediately after isolation from normal rats, but slight in hepatic stellate cells assumed nearly quiescent in function after 3 days of culture on plastic dishes. When rat received carbon tetrachloride, liver necrosis developed between 1 and 3 days following the intoxication. In these rats, osteopontin mRNA expression assessed by quantitative competitive RT-PCR was increased in the liver later than 1 day with its peak at 2 days following the intoxication. Kupffer cells and hepatic macrophages and hepatic stellate cells isolated from such liver showed marked expression of osteopontin mRNA on Northern blotting. Immunohistochemical examination disclosed that osteopontin was stained in macrophages including Kupffer cells and stellate cells in the necrotic areas. On electron microscopy, osteopontin stains were present in the Golgi apparatus in these cells. Recombinant human osteopontin promoted migration of Kupffer cells isolated from normal rats and cultured in a Transwell cell culture chamber in a dose-related manner. We conclude that activated Kupffer cells and hepatic macrophages and stellate cells express osteopontin. These cells might contribute to the infiltration of Kupffer cells and macrophages into hepatic necrotic areas by expressing osteopontin.

Animals↗
PubMed1999

Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis.

Vascular endothelial growth factor (VEGF) can induce proliferation of sinusoidal endothelial cells. Its mRNA expression was increased in proliferating rat hepatocytes in primary culture. To clarify a role of VEGF in liver after necrosis, expressions of VEGF and its receptors were measured in the liver or liver cells isolated from rats after carbon tetrachloride intoxication. Hepatic VEGF mRNA expression increased later than 24 h after the intoxication and became prominent at 168 h when liver necrosis disappeared, while hepatic mRNA expressions of its receptors increased between 24 and 72 h. VEGF mRNA expression was increased in Kupffer cells, hepatic macrophages and stellate cells isolated from rats between 24 and 72 h after the intoxication and in hepatocytes at 168 h compared to those cells from normal rats. Immunohistochemical VEGF stains were comparable to such results. Vascular endothelial cells existed abundantly in the necrotic areas, and sinusoidal endothelial cells appeared following disappearance of the necrotic areas. VEGF mRNA expression in hepatocytes isolated from 70% resected liver was increased at 12 h after the operation and became marked between 72 and 168 h. Similar increase of hepatic VEGF expression was immunohistochemically seen. In conclusion, VEGF derives from nonparenchymal as well as parenchymal cells in rat liver after necrosis. The former might contribute to vascular endothelial cell proliferation and the latter to sinusoidal endothelial cell regeneration.

Animals↗
PubMed1999

Deranged blood coagulation equilibrium as a factor of massive liver necrosis following endotoxin administration in partially hepatectomized rats.

Activated Kupffer cells provoke massive liver necrosis after endotoxin stimulation through microcirculatory disturbance caused by sinusoidal fibrin deposition in rats undergoing 70% hepatectomy. In these rats, serum activities of purine nucleoside phosphorylase (PNP) and alanine transaminase (ALT) were increased at 1 and 5 hours, respectively, following endotoxin administration. When 70% resected liver was perfused with Dulbecco's modified Eagle medium (DMEM) containing heat-inactivated fetal calf serum, the increase in both enzyme activities was not affected by addition of endotoxin during perfusion, suggesting that activated Kupffer cells injured neither sinusoidal endothelial cells nor hepatocytes. The activity of tissue factor, an initiator of blood coagulation cascade, was much higher in Kupffer cells isolated from partially hepatectomized rats than in those from normal rats. In contrast, mRNA expressions of tissue factor pathway inhibitor (TFPI) as well as thrombomodulin were almost undetectable in normal and partially resected livers. When recombinant human TFPI was injected intravenously in 70% hepatectomized rats, TFPI was markedly stained on the surfaces of sinusoidal endothelial cells and microvilli of hepatocytes on immunohistochemistry. In these rats, endotoxin-induced liver injury was significantly attenuated compared with rats given no TFPI. Similar attenuation was also found in rats receiving recombinant human thrombomodulin. These results suggest that fibrin deposition developing in 70% hepatectomized rats after endotoxin administration may be caused by deranged blood coagulation in the hepatic sinusoids through increasing tissue factor activity in Kupffer cells and minimal TFPI and thrombomodulin in endothelial cells. The destruction of sinusoidal endothelial cells as well as hepatocytes may occur as a result of microcirculatory disturbance caused by such sinusoidal fibrin deposition.

Alanine Transaminase↗