Decreased expression of receptors for vascular endothelial growth factor and sinusoidal endothelial cell damage in cold-preserved rat livers.
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Animals↗
Biomedical subjects
S Mochida
Publications and source records attributed to S Mochida.
Snapin: a SNARE-associated protein implicated in synaptic transmission.
Synaptic vesicle docking and fusion are mediated by the assembly of a stable SNARE core complex of proteins, which include the synaptic vesicle membrane protein VAMP/synaptobrevin and the plasmalemmal proteins syntaxin and SNAP-25. We have now identified another SNAP-25-binding protein, called Snapin. Snapin was enriched in neurons and exclusively located on synaptic vesicle membranes. It associated with the SNARE complex through direct interaction with SNAP-25. Binding of recombinant Snapin-CT to SNAP-25 blocked the association of the SNARE complex with synaptotagmin. Introduction of Snapin-CT and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons in culture reversibly inhibited synaptic transmission. These results suggest that Snapin is an important component of the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin.
Amino Acid Sequence↗
Minor triterpenoid saponins from Ardisia crenata.
Two minor triterpenoid saponins, ardisicrenoside G [3 beta-O-{alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)- [beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl}-16 alpha,28-dihydroxyolean-12-en-30-oic acid] and ardisicrenoside H [3 beta-O-{beta-D-xylopyranosyl-(1-->2)-beta-D- glucopyranosyl-(1-->4)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L- arabinopyranosyl}-16 alpha,28-dihydroxyolean-12-en-30-oic acid] were isolated from the roots of Ardisia crenata. Structural assignments are based on NMR, MS and chemical reactions.
Carbohydrate Sequence↗
Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels.
Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their alpha1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein-protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.
Action Potentials↗
Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process.
Doc2alpha and Munc13-1 proteins are highly concentrated on synaptic vesicles and the presynaptic plasma membrane, respectively, and have been implicated in Ca2+-dependent neurotransmitter release. Doc2alpha interacts with Munc13-1 through the N-terminal region of Doc2alpha (the Mid domain; amino acid residues 13-37). Here we examine whether the interaction between Doc2alpha and Munc13-1 is required for Ca2+-dependent neurotransmitter release from intact neuron. A synthetic Mid peptide (the Mid peptide), but not a control mutated Mid peptide or a scrambled Mid peptide, inhibited the interaction between Doc2alpha and Munc13-1 in vitro. Introduction of the Mid peptide into presynaptic neurons of cholinergic synapses, formed between rat superior cervical ganglion neurons, reversibly inhibited synaptic transmission evoked by action potentials. In contrast, the control peptides did not inhibit synaptic transmission. This inhibitory effect depended on the presynaptic activity and was affected by extracellular Ca2+ concentrations. The onset of the Mid peptide effect was shortened when the neuron was stimulated at a higher frequency, and the inhibition was more potent at 1 mM Ca2+ than at 5.1 mM Ca2+. These results suggest that the Doc2alpha-Munc13-1 interaction plays a role in a step before the final fusion step of synaptic vesicles with the presynaptic plasma membrane in the evoked neurotransmitter release process.
Animals↗
Contribution of CD14 to endotoxin-induced liver injury may depend on types of macrophage activation in rats.
Activated Kupffer cells and hepatic macrophages can produce massive liver necrosis through microcirculatory disturbance due to sinusoidal fibrin deposition. This mechanism is involved in the development of liver injury after endotoxin administration in rats pretreated with heat-killed Propionibacterium acnes (P.acnes) or undergoing 70% liver resection. The significance of CD14, a receptor for lipopolysaccharide and its binding protein, was evaluated in both models in relation to the activation mechanisms of Kupffer cells and hepatic macrophages. Northern blot analysis revealed that CD14 mRNA expression was increased in the liver of rats following P.acnes administration. In these rats, hepatic macrophages immediately after isolation showed marked increased of CD14 mRNA expression compared to Kupffer cells from normal rats. In contrast, CD14 mRNA expression was minimal in partially resected liver. Interleukin (IL)-18 and IL-2 mRNA expression in the liver and interferon (IFN)-gamma mRNA expression in the spleen were significantly increased in P.acnes-treated rats compared to normal rats, while these increases were absent in partially hepatectomized rats. Thus, CD14 expressed on hepatic macrophages after activation through a cytokine network of IL-18, IFN-gamma, and IL-2 may contribute to endotoxin-induced liver injury in P.acnes-treated rats. In contrast, in partially hepatectomized rats, this network may not operate during Kupffer cell activation, and the liver injury might develop through endotoxin receptors other than CD14 on the cells.
Animals↗
Selective bowel decontamination of recipients for prevention against liver injury following orthotopic liver transplantation: evaluation with rat models.
Gut-derived substances can activate Kupffer cells to provoke hepatic necrosis after partial hepatectomy in rats. A similar situation may occur during orthotopic liver transplantation (OLT), as congestion in the intestinal wall, caused by portal vein occlusion, is inevitable during the operation. The contribution of such substances to liver injury following OLT was investigated in rats. Oral administration of polymyxin B sulfate for 7 days significantly altered intestinal bacterial flora in rats; Enterobacteriaceae diminished and anaerobes such as Bifidobacterium , Lactobacillus, Bacteroides, and Eubacterium increased in number, compared with the control rats. Also, this treatment significantly reduced endotoxin concentration in the portal blood 30 minutes after blood reflow following portal vein occlusion. When OLT was performed in rats using the liver preserved in cold University of Wisconsin solution for 18 hours, tissue factor activity in Kupffer cells (KC) isolated from the transplanted liver 1 hour after the operation was significantly higher than in that of normal rats. This increase was significantly reduced by pretreatment of the recipients with polymyxin B sulfate. In these recipients, serum alanine aminotransferase activity, tumor necrosis factor alpha (TNF alpha) concentration, and histological extent of liver necrosis were significantly attenuated at 24 hours after the operation compared with those of control rats. We conclude that the substances derived from bacilli sensitive to polymyxin B sulfate in the gut may be a contributing factor to liver injury following OLT in rats; we feel that this probably occurs by entering of the substances into the portal blood during the ahepatic phase of the operation to activate KC. Selective bowel decontamination of recipients with polymyxin B sulfate would be a candidate for protection against early graft failure following OLT.
Animals↗
Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins.
Clostridium botulinum type B neurotoxin (BoNT/B) recognizes a complex of synaptotagmin II and ganglioside GT1b or GD1a as the high-affinity toxin binding site. Recombinant deletion mutants of synaptotagmin II allowed us to demonstrate that the N-terminal domain including the transmembrane region retains BoNT/B binding activity while the C-terminal domain is not involved in constituting the BoNT/B receptor. BoNT/B binding to reconstituted lipid vesicles containing synaptotagmin II and gangliosides showed that GT1b and GD1a confer the difference in the maximum binding capacity but not in the dissociation constant. The direct binding of GT1b to the deletion mutants revealed that the transmembrane region is required to bind GT1b, suggesting that synaptotagmin II binds to the ceramide portion of gangliosides within the plasma membrane. A monoclonal antibody against GT1b effectively inhibited not only BoNT/B binding to the reconstituted lipid vesicles and brain synaptosomes but also type A BoNT (BoNT/A) binding to brain synaptosomes. In addition, the monoclonal antibody antagonized the action of both BoNT/A and BoNT/B on synaptic transmission of rat superior cervical ganglion neurons. These results suggest that GT1b functions as a component of the receptor complex.
Animals↗
Anticoagulant targeting for hepatic sinusoidal walls in prevention of hypercoagulopathy in cold preserved rat livers.
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Animals↗
Enhancement of albumin production by hepatocyte growth factor in rat hepatocytes: distinction in mode of action from stimulation of DNA synthesis.
Hepatocyte growth factor (HGF) has been shown to enhance albumin production as well as stimulate DNA synthesis in hepatocytes. The mode of action of HGF in exerting both effects is to be elucidated. We previously observed that hepatocyte proliferation occurred in normal rats given recombinant human HGF (rhHGF) intravenously. When rats received rhHGF similarly, serum albumin levels were significantly increased compared to controls. In primary culture of rat hepatocytes, albumin concentration in culture medium was significantly increased by rhHGF added at 24 h of culture compared to controls, increasing with time of culture. This effect of rhHGF was dose-related. When actinomycin D was added to the medium, the albumin concentration was reduced in a dose-related manner, but its enhancement by rhHGF was maintained. Albumin mRNA levels were not increased by rhHGF. When rhHGF was added similarly to the medium, immunocytochemically positive hepatocytes for 5-bromo-2'-deoxyuridine (BrdU) incorporation appeared 30 h later. Of these labeled hepatocytes, 12.5% were concomitantly stained for albumin. In contrast, albumin-positive hepatocytes were seen in 77% of BrdU-non-labeled hepatocytes (p<0.01). We conclude that HGF may enhance albumin production through post-transcriptional regulation in non-proliferating hepatocytes, but not in proliferating hepatocytes.
Animals↗
The mechanisms of hepatic sinusoidal endothelial cell regeneration: a possible communication system associated with vascular endothelial growth factor in liver cells.
Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.
Animals↗
Effect of FK506 on the activation state of hepatic macrophages in Propionibacterium acnes-treated rats.
Activated hepatic macrophages can provoke massive liver necrosis following endotoxin stimulation through microcirculatory disturbances due to sinusoidal fibrin deposition in rats pretreated with heat-killed Propionibacterium acnes. In these rats, FK506 (tachlorinus) administered 24 h before and at the time of endotoxin injection, significantly attenuated liver injury compared with the rats given no FK506. The effect of FK506 on hepatic macrophage activation and its action sites were studied in Propionibacterium acnes-treated rats. When rats received Propionibacterium acnes intravenously, hepatic-mRNA expression of interferon-gamma-inducing factor and interleukin-2 and splenic-mRNA expression of interferon-gamma were significantly increased compared with normal rats. Hepatic-mRNA expression of CD14, a receptor for lipopolysaccharide and its binding protein complex, was also increased preceding the expressions of the three cytokines in the liver and spleen. FK506 administration attenuated hepatic-mRNA expression of interleukin-2 and both superoxide anions as well as tumour necrosis factor-alpha production by hepatic macrophages, but did not change CD14-mRNA expression in Propionibacterium acnes-treated rats. It is suggested that a cytokine network through interferon-gamma-inducing factor, interferon-gamma and interleukin-2 may operate during activation of hepatic macrophages in rats treated with heat-killed Propionibacterium acnes, while CD14 expression on the cells may increase independently of this network. FK506 seems to attenuate such activation by suppressing hepatic interleukin-2 expression, without affecting CD14 expression on the cells.
Animals↗
Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1.
Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.
Amino Acid Sequence↗
Recombinant human tissue factor pathway inhibitor as a possible anticoagulant targeting hepatic sinusoidal walls.
The expression of tissue factor pathway inhibitor (TFPI) was investigated in sinusoidal endothelial cells in the liver and endothelial cells in the lung. Northern blot analysis revealed that TFPI mRNA was expressed in the lung, but minimal in the liver. Also, immunohistochemical examination showed that TFPI was not expressed on the sinusoidal endothelial cells in contrast to marked expression on endothelial cells in the lung, suggesting that anticoagulant activity to inhibit blood coagulation induced by tissue factor is reduced in the hepatic sinusoids compared to the microvessels of other organs. When recombinant human TFPI was intravenously injected in rats, it disappeared rapidly from the circulation, but was detected by electron microscopy on the surface of sinusoidal endothelial cells and microvilli of hepatocytes in the space of Disse. In these rats, the TFPI reappeared in the circulation following an intravenous injection of heparin sodium with reduced immunohistochemical staining of the TFPI on hepatic sinusoidal walls. It is concluded that exogenous TFPI can increase anticoagulant activity on the hepatic sinusoidal walls by binding to heparinoids on the cell surface. It may act effectively even in the hepatic sinusoids with damaged endothelial cells.
Animals↗
Pitfall in the implication of intercellular adhesion molecule-1 expression on isolated hepatocytes.
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Animals↗
Effects of neurotropic pyrimidine heterocyclic compound, MS-430, on cultured hepatic parenchymal and stellate cells.
MS-430 is a novel synthetic pyrimidine derivative that stimulates regeneration of the nerve as a promoter for various growth factors such as epidermal growth factor (EGF) and nerve growth factor, and differentiation of astrocytes. The effects of MS-430 on the liver were tested using hepatocytes and stellate cells in primary culture isolated from rats. MS-430 enhanced EGF-induced DNA synthesis in hepatocytes while it alone failed to increase the basal DNA synthesis. Albumin mRNA expression in the cells and its amount in the medium were not changed by addition of EGF or MS-430 alone or both. Basic fibroblast growth factor (bFGF) increased DNA and but not collagen synthesis by hepatic stellate cells. Addition of MS-430 inhibited DNA synthesis by hepatic stellate cells at either presence or absence of bFGF, and collagen synthesis at the presence of bFGF. However, MS-430 had no effects on basal or bFGF-stimulated TGFbeta mRNA expression in the cells. These results suggest that MS-430 stimulated proliferation of hepatocytes as a comitogen for EGF without affecting albumin synthesis, and suppressed proliferation of activated hepatic stellate cells and their collagen synthesis without affecting TGFbeta expression.
Animals↗
Roles of synaptotagmin C2 domains in neurotransmitter secretion and inositol high-polyphosphate binding at mammalian cholinergic synapses.
To determine the functional role of synaptotagmin (Syt) regulatory domains, affinity-purified antibodies specific for C2A or C2B domains were injected into presynaptic neurons of cholinergic synapses formed between rat sympathetic neurons in culture. Following injection of anti-C2A antibody, postsynaptic responses evoked by presynaptic action potentials at a frequency of 0.05 Hz decreased rapidly, while anti-C2B antibody slowly decreased synaptic transmitter release. The inhibitory effect of anti-C2B antibody depended on the amount of synaptic activity. Asynchronous release induced by hypertonic solution was also affected by the antibodies. Anti-C2A antibody showed a dual action on miniature excitatory postsynaptic potentials, a decrease and following increase in the frequency, while synapses loaded with anti-C2B antibody showed a decrease in the frequency after long repetitive stimulation (0.05 Hz for more than 60 min). Anti-C2B antibody prevented the inhibition of acetylcholine release induced by injection of inositol 1,3,4,5-tetrakisphosphate (IP4), indicating that C2B domain may down-regulate transmitter release by IP4 binding. These results confirm similar experiments in the glutamatergic squid giant synapses and suggest a model in which Syt C2A and C2B domains differentially control synaptic vesicle trafficking in mammalian cholinergic terminals; C2A domain may act on the fusion step as a calcium sensor in synaptic vesicle exocytosis evoked by action potentials in addition to controlling spontaneous transmitter release, while C2B domain is involved in exo- and endocytosis.
Acetylcholine↗
Blood coagulation in the hepatic sinusoids as a contributing factor in liver injury following orthotopic liver transplantation in the rat.
Blood coagulation equilibrium in the hepatic sinusoids may be deranged after orthotopic liver transplantation, since tissue factor activity increases in Kupffer cells and thrombomodulin expression disappears in sinusoidal endothelial cells in orthotopically transplanted rat liver. The presence of sinusoidal blood coagulopathy and its contribution to the development of early graft failure following orthotopic liver transplantation were investigated in rats. Orthotopic liver transplantation was performed using livers preserved in cold UW solution, according to the method of Kamada, in rats. Abnormalities in the hepatic sinusoids were evaluated electron-microscopically. Anticoagulation therapy was done using antithrombin III concentrate infused immediately and 12 hr after operation. Fibrin deposition and endothelial cell destruction in the hepatic sinusoids were observed in transplanted rats 5 hr after operation. In these rats, plasma antithrombin III activity was decreased to less than 40%o of normal levels, and massive hepatic necrosis developed with increased plasma thrombin-antithrombin III complex concentration 24 hr after operation. Anticoagulation therapy significantly attenuated the extent of hepatic necrosis, with normalization of plasma antithrombin III activity and further increase in plasma thrombin-antithrombin III complex concentration. A hypercoagulative state exists after orthotopic liver transplantation in rats. Fibrin deposition in the hepatic sinusoids associated with this state may contribute to the development of early graft injury.
Animals↗