[Inhibition of neurotransmitter release by tetanus and botulinum neurotoxins].
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Amino Acid Sequence↗
Biomedical subjects
S Mochida
Publications and source records attributed to S Mochida.
Coagulability in the sinusoids of orthotopically transplanted livers in rats.
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Adenosine↗
Sinusoidal endothelial cell damage by activated macrophages in rat liver necrosis.
BACKGROUND: Massive hepatic necrosis caused by fibrin deposition in the hepatic sinusoids develops with hepatic macrophage activation in rats given endotoxin after administration of heat-killed Corynebacterium parvum. Targeted cells of such macrophages were investigated. METHODS: In C. parvum-treated rats, the pathological appearance of liver cells was serially measured in serum following endotoxin administration and compared with the appearance in the perfusate during closed liver perfusion with endotoxin. RESULTS: Serum activities of tumor necrosis factor, purine nucleoside phosphorylase present in both hepatocytes and sinusoidal endothelial cells, and levels of alanine aminotransferase were higher after 30 minutes, 1 hour, and 3 hours, respectively. Pretreatment of rats with gadolinium chloride, an inhibitor of macrophage function, reduced this liver injury. Although alanine aminotransferase activity remained almost unchanged in the liver perfusate, purine nucleoside phosphorylase activity increased. This increase was reduced when rats were pretreated with gadolinium chloride. There was sinusoidal endothelial cell damage around hepatic macrophages in the liver perfused with endotoxin. CONCLUSIONS: Activated hepatic macrophages may cause sinusoidal endothelial cell damage leading to hepatocyte necrosis in rats given C. parvum and endotoxin.
Alanine Transaminase↗
Stimulation of liver growth by exogenous human hepatocyte growth factor in normal and partially hepatectomized rats.
Human hepatocyte growth factor stimulates DNA synthesis by cultured rat hepatocytes. When human hepatocyte growth factor prepared from the culture medium of human embryonic lung fibroblasts was intravenously injected into normal rats and rats after 70% hepatectomy, it was detected in hepatocytes but not in nonparenchymal cells isolated 30 min after injection. Similar injections of human hepatocyte growth factor at 2-hr intervals for 10 hr significantly increased hepatic DNA content in normal rats at 48 hr, with increased hepatic content of putrescine, the essential polyamine for hepatic DNA synthesis after 70% hepatectomy, and activities of catalytic enzymes of putrescine synthesis at 6 hr almost to the levels in rats after 70% hepatectomy. Those levels in rats after 70% hepatectomy were further enhanced by similar injections of human hepatocyte growth factor starting immediately after surgery. Increased hepatic DNA content in normal rats and rats after 70% hepatectomy was also seen with recombinant human hepatocyte growth factor to a greater extent compared with that seen with human hepatocyte growth factor. In normal rats given recombinant human hepatocyte growth factor, 5-bromo-2'-deoxyuridine-labeled and mitotic hepatocytes were significantly increased in number at 26 hr but not at 48 hr. We conclude that exogenous human hepatocyte growth factor acts as a trigger and a promoter of liver growth to increase hepatic putrescine production in rats. Recombinant human hepatocyte growth factor is more potent than human hepatocyte growth factor in this action.
Animals↗
Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A.
To define conserved domains within the light (L) chains of clostridial neurotoxins, we determined the sequence of botulinum neurotoxin type B (BoNT/B) and aligned it with those of tetanus toxin (TeTx) and BoNT/A, BoNT/C1, BoNT/D, and BoNT/E. The L chains of BoNT/B and TeTx share 51.6% identical amino acid residues whereas the degree of identity to other clostridial neurotoxins does not exceed 36.5%. Each of the L chains contains a conserved motif, HExxHxxH, characteristic for metalloproteases. We then generated specific 5'- and 3'-deletion mutants of the L chain genes of TeTx and BoNT/A and tested the biological properties of the gene products by microinjection of the corresponding mRNAs into identified presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Toxicity was determined by measurement of neurotransmitter release, as detected by depression of postsynaptic responses to presynaptic stimuli (Mochida, S., Poulain, B., Eisel, U., Binz, T., Kurazono, H., Niemann, H., and Tauc, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7844-7848). Our studies allow the following conclusions. 1) Residues Cys439 of TeTx and Cys430 of BoNT/A, both of which participate in the interchain disulfide bond, play no role in the toxification reaction. 2) Derivatives of TeTx that lacked either 8 amino- or 65 carboxyl-terminal residues are still toxic, whereas those lacking 10 amino- or 68 carboxyl-terminal residues are nontoxic. 3) For BoNT/A, toxicity could be demonstrated only in the presence of added nontoxic heavy (H) chain. A deletion of 8 amino-terminal or 32 carboxyl-terminal residues from the L chain had no effect on toxicity, whereas a removal of 10 amino-terminal or 57 carboxyl-terminal amino acids abolished toxicity. 4) The synergistic effect mediated by the H chain is linked to the carboxyl-terminal portion of the H chain, as demonstrated by injection of HC-specific mRNA into neurons containing the L chain. This finding suggests that the HC domain of the H chain becomes exposed to the cytosol during or after the putative translocation step of the L chain.
Acetylcholine↗
In situ detection of oxidative stress in rat hepatocytes.
In rat hepatocytes in primary culture incubated with nitro blue tetrazolium, formazan content was increased by addition of t-butyl hydroperoxide, a potent oxidant, in a dose-related manner, but not by addition of valinomycin, which kills hepatocytes through mitochondrial damage. This increment after t-butyl hydroperoxide addition was not seen in hepatocytes preincubated with deferoxamine mesylate, a ferric iron chelator which inhibits radical formation. Liver perfusion with nitro blue tetrazolium and t-butyl hydroperoxide in rats produced formazan deposition faintly on the surface of hepatocytes throughout the liver and prominently in the cytoplasm of some hepatocytes, which was attenuated when performed following deferoxamine mesylate perfusion. When liver perfusion with nitro blue tetrazolium was performed in carbon tetrachloride-intoxicated rats, formazan deposition appeared diffusely in hepatocytes in the centrilobular areas. Similar deposition was also observed on the surface and in the cytoplasm of hepatocytes in the periportal and mid-zonal areas in rats undergoing post-ischaemic reperfusion. Liver perfusion with nitro blue tetrazolium can detect in situ oxidative stress in hepatocytes and may be a useful tool for studying the role of lipid peroxidation in rat liver injury.
Animals↗
Hepatocyte membrane stabilization by prostaglandins E1 and E2: favorable effects on rat liver injury.
When prostaglandin (PG) E1 was continuously administered to rats from 24 hours before giving a dose of carbon tetrachloride, deranged serum glutamic pyruvic transaminase levels and prothrombin time were significantly reduced 12 hours after intoxication compared with controls. A similar effect of PGE1 was seen at 24 hours in D-galactosamine-intoxicated rats. Liver histology showed a comparable attenuation of injury in these rats. These results were consistent with reported effects of PGE2, suggesting that both prostaglandins may share a common pathway in protection against liver injury. When PGE1 or 16,16'-dimethyl PGE2 was added to the medium of primary cultured rat hepatocytes, lipid peroxidation-dependent killing of the cells by tert-butyl hydroperoxide was significantly attenuated without affecting the extent of malondialdehyde accumulation compared with controls. Both prostaglandins significantly reduced the extent of increased plasma membrane microviscosity of these cells assessed by 1-[4-(trimethyl-ammonio)phenyl]-6-phenyl-1,3,5-hexatriene. PGE1 and PGE2 may possess cytoprotective effects on liver parenchymal cells through stabilization of membrane microviscosity, which may contribute to protection against liver injury.
16,16-Dimethylprostaglandin E2↗
Gut-derived substances in activation of hepatic macrophages after partial hepatectomy in rats.
When liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was performed in rats 24 h after two-thirds liver resection, there were marked deposits of formazan converted from nitro blue tetrazolium in hepatic macrophages throughout the liver, indicating macrophage activity. The extent of the deposits was significantly reduced when perfusion was performed following oral administration of polymyxin B sulfate, a non-absorbable bacteriocidal agent against gram-negative bacilli which can also bind endotoxin lipopolysaccharides. Polymyxin B sulfate administration also attenuated the derangements of SGPT and the histological liver injury provoked by endotoxin administration after partial hepatectomy. These results suggests that gut-derived substances sensitive to polymyxin B sulfate may contribute to activation of hepatic macrophages after partial hepatectomy in rats.
Animals↗
HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri.
An aqueous extract of Phyllanthus niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 reverse transcriptase (HIV-1-RT). The inhibitor against HIV-1-RT in this plant was purified by combination of three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus repandus. The 50% inhibitory doses (ID50) of RA on HIV-1-RT and DNA polymerase alpha (from HeLa cells) were 0.05 microM and 0.6 microM, respectively, representing approximately a 10-fold more sensitivity of HIV-1-RT compared with DNA polymerase alpha. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 microM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 microM of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 microM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system.
Antigens, Viral↗
Intracellular transduction mechanisms for the slow synaptic events.
Electrical activities of the postganglionic neurons in the superior cervical ganglia of rabbits are modulated in various ways following activation of the subtypes of muscarinic acetylcholine receptors. (i) M1 receptors mediate a slow depolarization consisting of at least three types of ionic conductance changes, and one of these is possibly mediated by cyclic GMP. (ii) M2 receptors mediate a slow hyperpolarization that seems to be generated by inositol triphosphate derived from phosphatidylinositol breakdown. (iii) M2 receptors also cause, through an activation of C kinase, a suppression of Ca entry during action potentials that results in a characteristic change in the action potentials and thereby modulates excitability of superior cervical ganglion neurons. Each subtype of muscarinic receptors thus regulates different pathways of intracellular transduction and modulates the electrical signaling of sympathetic neurons.
Animals↗
Rapid reconstruction of three dimensional images from ultrasonography using a linearly moving mirror.
A system for reconstructing three dimensional (3-D) images from ultrasonography using a linearly moving mirror was modified. The device could display the 3-D structure of objects in real time by employing an on-line system. In a case of hepatocellular carcinoma, structures of the tumor and blood vessels were selectively visualized. One 3-D image was generated within five minutes. This modified system will be of use in the analysis of liver tumors.
Aged↗
Heterologous combinations of heavy and light chains from botulinum neurotoxin A and tetanus toxin inhibit neurotransmitter release in Aplysia.
The neuroparalytic activities of botulinum neurotoxin type A (BoNT A), tetanus toxin (TeTx), or homologous and heterologous combinations of their constituent polypeptides were examined at cholinergic and non-cholinergic synapses of Aplysia californica. When applied extracellularly, BoNT A or a mixture of its heavy (HC) and light (LC) chains were far more potent in blocking transmitter release at cholinergic than non-cholinergic synapses. The reverse was true for TeTx or a mixture its constituent chains. Such selectivity was assigned to differences in neuronal targetting and uptake of the neurotoxins since both exhibited similar potencies when injected directly into the cell body of either cell type. When bath-applied, heterologous combinations of the toxins' HC and LC appeared as effective as the parent neurotoxins from whence each HC was derived. Moreover, targetting/internalization was attributable to the analogous N-terminal moieties, H2 and beta 2, of the HC from BoNT A and TeTx. Thus, it may be postulated that the latter regions possess two functional domains, one being distinct and responsible for the divergent neuronal specificity, whereas the other serves a common role in translocating the LC of either toxin. Also, it was shown that the C-terminal portion of the HC of TeTx is unable to play the intracellular role of its counterpart in BoNT A.
Animals↗
Ruffle formation in the evaluation of stimulatory state of hepatic macrophages in rats.
Morphological changes in hepatic macrophages after stimulation were observed with a transmission electron microscope in rats. In the normal liver ruffle formation occupied less than 30% of the cell surface facing the sinusoidal space in 21 of 25 macrophages. When the rats received a dose of carbon tetrachloride, hepatic macrophages 1 day later showed ruffle formation to the same extent as in normal rats. In contrast, in rats 6 days after a dose of Corynebacterium parvum, ruffle formation was intensified, and in 19 of 25 macrophages it was seen to occupy more than 30% of the cell surface (p less than 0.01); 9 of 10 macrophages with more than 70% of the cell surface affected belonged to these rats. Considering that hepatic macrophages at 1 or 6 days after treatment with carbon tetrachloride or Corynebacterium parvum are at the responsive or primed stages, respectively, measurement of the extent of ruffle formation in hepatic macrophages with a transmission electron microscope may provide a useful tool in estimating their stimulatory stage.
Animals↗
Evidence for enhanced secretory function of hepatic macrophages after long-term ethanol feeding in rats.
Rats were pair-fed nutritionally adequate liquid diets, containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4-6 weeks. Ruffle formation of hepatic macrophages in the periportal area observed with a transmission electron microscope (which reflects their extent in activation) was more remarkable in ethanol-fed rats than in control rats. The ability of hepatic macrophages to produce superoxide anions assessed in situ by formazan deposition after liver perfusion with nitro-blue tetrazolium and phorbol myristate acetate was enhanced after such ethanol feeding. A similar result was seen 24 h after withdrawal of ethanol feeding. These findings suggest that long-term ethanol consumption may activate hepatic macrophages in secretory function.
Animals↗
Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria.
Rts1 is a multiphenotype drug resistance factor, and one of its phenotypes is temperature-sensitive growth (Tsg) of host bacteria. A 3.65-kb fragment from Rts1 DNA was shown to cause the Tsg phenotype in host cells. This tsg fragment was split by a restriction enzyme, HincII, into four fragments. Two of these fragments were called HincII-S (short) and HincII-L (long), respectively. Each of these two fragments conferred the Tsg phenotype, indicating that, in fact, these two independent regions were responsible for the Tsg phenotype. The HincII-S 783-bp and HincII-L 1,479-bp fragments were sequenced. The region in the HincII-S fragment to which the Tsg phenotype was attributed was narrowed to a 146-bp (nucleotides 1 to 146) fragment by various restriction enzyme digestions. Further digestion of the 146-bp fragment with Bal 31 suggested that the 116-bp (nucleotides 9 to 124) fragment is the minimum sequence required for Tsg. On the other hand, in the HincII-L fragment, a fragment of 249 bp (nucleotides 1210 to 1458) and a fragment of 321 bp (nucleotides 1942 to 2262) contained separate temperature-sensitive growth activity. None of three tsg fragments contained open reading frames. The 249-bp fragment had very weak Tsg activity, while the 321-bp fragment had no Tsg activity. On the other hand, when these two fragments were together in the pUC19 vector, they exhibited very strong Tsg activity equivalent to that of the original 1,479-bp fragment. In addition, two of the 249-bp fragments gave similar, strong Tsg activity. The HincII-L 1,479-bp fragment contained an open reading frame for kanamycin resistance which was found between nucleotides 1423 and 2238. This kanamycin resistance gene sequence was different from that of the reported kanamycin resistance gene of Tn903 at 12 positions which were deduced to change seven amino acids.
Base Sequence↗
Method for in situ evaluation of superoxide production by pulmonary macrophages in the rat.
In order to investigate superoxide production by pulmonary macrophages in the rat, a route was created by ligating both the inferior and superior venae cavae and resecting the aorta after cannulation through the inferior vena cava into the right atrium of the heart. Lung perfusion was performed via this route with nitro blue tetrazolium. Although there was no formazan deposition throughout the lung, it became detectable in both alveolar and interstitial macrophages when phorbol myristate acetate was added to the perfusate. This deposition was markedly enhanced by previous injection of Corynebacterium parvum. The deposition disappeared after further addition of Cu(Lys)2, a scavenger of superoxide anions. This procedure may be useful for estimating in situ the ability of pulmonary macrophages to produce superoxide in the rat.
Animals↗
Usefulness of pulsed Doppler ultrasound in detection of angiographically evident recurrence of hepatocellular carcinoma after arterial embolization treatment.
Because hepatocellular carcinoma treated by transcatheter arterial embolization often regains its size, routine follow-up is necessary. The usefulness of pulsed Doppler ultrasound for detection of this type of recurrence was compared with ultrasonography and computed tomography in 21 such hepatocellular carcinomas. Of 15 hepatocellular carcinomas diagnosed by angiography as showing recurrence, four were detected with ultrasonography and five were detected with computed tomography. Doppler signals were obtained in the peripheral portions corresponding to tumor vessels or stains on angiograms in 14 of these 15 hepatocellular carcinomas, but they were undetectable in six hepatocellular carcinomas with no recurrence. All signals disappeared after transcatheter arterial embolization. One false-negative hepatocellular carcinoma with pulsed Doppler ultrasound showed faint tumor stains on angiograms; these were also negative on ultrasonography and computed tomography. Pulsed Doppler ultrasound may be superior to ultrasonography and computed tomography as a routine procedure to detect the recurrence of hepatocellular carcinoma treated by transcatheter arterial embolization.
Aged↗
Minor contribution of hepatocytes to collagen production in normal and early fibrotic rat livers.
Hepatocyte contribution to hepatic collagen production in vivo was estimated in rats, based on the fact that ornithine is used for protein synthesis in the liver as arginine after conversion by way of the urea cycle only by hepatocytes. From rats given a mixture of [14C] ornithine and [3H]arginine, hepatic collagen and serum albumin were obtained. The hepatocyte contribution was calculated from the 14C and 3H in arginine purified from collagen and albumin by high performance liquid chromatography. The contribution was less than 10% of total collagen production in normal and early fibrotic livers induced by a single dose of carbon tetrachloride or dimethylnitrosamine. We conclude that hepatocytes may play a minor role in collagen production in normal and early fibrotic rat livers.
Amino Acids↗