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Biomedical subjects

S Yamaguchi

Publications and source records attributed to S Yamaguchi.

At least 19 recordsLinked to original sources

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.

Bacterial Proteins

Localization of the Salmonella typhimurium flagellar switch protein FliG to the cytoplasmic M-ring face of the basal body.

The direction of rotation of the bacterial flagellum is determined by the flagellar switch. We have localized FliG, one of the switch proteins of Salmonella typhimurium, to the cytoplasmic face of the M ring of the flagellar basal body. This localization was made possible by the discovery of two spontaneous mutants in which the fliF (M ring) and fliG (switch) genes were fused in-frame. In the first mutant, a deletion of 7 base pairs at the 3' end of fliF resulted in an essentially full-length fusion protein. In the second mutant, a larger deletion resulted in a fusion in which 56 amino acids from the carboxyl terminus of FliF and 94 amino acids from the amino terminus of FliG were lost. Both strains were motile and underwent switching; the first strain had a clockwise bias, and the second strain had a counterclockwise bias. Gel electrophoresis and immunoblotting of isolated hook-basal-body complexes verified that they contained the fusion proteins. Electron microscopy revealed additional mass at the cytoplasmic face of the M ring, which could be decorated with anti-FliG antibody. We conclude that the natural location for FliG is at the cytoplasmic face of the M ring and that the stoichiometric ratio between FliF and FliG in wild-type cells is probably 1:1.

Amino Acid Sequence

Molecular basis of 3-ketothiolase deficiency: identification of an AG to AC substitution at the splice acceptor site of intron 10 causing exon 11 skipping.

3-Ketothiolase deficiency (3KTD) is the result of a deficiency in mitochondrial acetoacetyl-CoA thiolase (T2). The molecular basis of 3KTD was analyzed in a patient (GK10) and his family at the protein, cDNA and gene levels. Protein analyses showed that GK10's T2 protein was undetectable in fibroblasts even with the pulse-protein labeling method and that his parents were carriers of 3KTD. Complementary DNA analyses with PCR showed that T2 cDNA in the patient lacked the normal exon 11 sequence and that his parents were obligatory carriers of the DNA sequence which canceled exon 11. When the PCR-amplified genomic fragments around exon 11 were sequenced, an AG to AC mutation at the 3' splice site of intron 10 was detected. This mutation is presumed to be responsible for exon 11 skipping.

Acetyl-CoA C-Acyltransferase

Protection against dendrotoxin-induced clonic seizures in mice by anticonvulsant drugs.

Various anticonvulsant drugs were evaluated for their ability to protect against clonic seizures induced in mice by intraventricular injection of the K+ channel blocking peptide dendrotoxin (DTX). Phenytoin, the phenytoin-like anticonvulsant carbamazepine and the broad spectrum drug valproate were effective in this model, whereas the GABA-enhancers diazepam and tiagabine, the NMDA antagonists (+/-)-CPP and (+)-MK-801, the AMPA antagonist NBQX, the antiabsence drug ethosuximide and the Ca2+ channel antagonist nimodipine were inactive. In contrast to the lack of activity of other NMDA antagonists, phencyclidine and ADCI [(+/-)-aminocarbonyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine] were potent antagonists of DTX-induced seizures.

Animals

Reversibility of catechol-induced rat glandular stomach lesions.

The potential reversibility of glandular stomach lesions induced by the clastogen, catechol, was examined in groups of male F344 rats treated continuously with 0.8% catechol in the diet for 12, 24, 48, 72, or 96 weeks. After a return to basal diet for 84, 72, 48, 24, and 0 weeks, respectively, the animals were killed for histopathological examination. Incidences of submucosal hyperplasia, adenomas and adenocarcinomas, average number of tumors per rat, and the size of tumors in rats treated with catechol for 12, 24, 48, 72, and 96 weeks increased time dependently. After cessation of catechol treatment, although average number of tumors per rat slightly decreased, the size of tumors tended to increase. Labeling indices in both tumorous and nontumorous areas decreased after cessation of catechol treatment. The results thus indicate that whereas some submucosal hyperplasias or adenomas may regress, others have the potential to develop into adenomas or adenocarcinomas. However, tumor growth does depend to a certain extent on continued catechol treatment.

Adenocarcinoma

Crystallization and characterization of monoacylglycerol and diacylglycerol lipase from Penicillium camembertii.

A new lipase from Penicillium camembertii U-150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalin-A-Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalin-A-Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. All four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase.

Amino Acids

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells.

Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1'-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.

Adrenoleukodystrophy

Identification of TaqI polymorphism in the mitochondrial acetoacetyl-CoA thiolase gene and familial analysis of 3-ketothiolase deficiency.

We analyzed the mitochondrial acetoacetyl-CoA thiolase gene (T2) by Southern blotting. Fifteen unrelated healthy individuals and members of five families with 3-ketothiolase deficiency (3KTD) were analyzed. We found a TaqI polymorphism, the heterozygosity of which was calculated to be 0.5 among healthy Japanese individuals. This restriction fragment length polymorphism (RFLP) proved to be useful for detecting 3KTD patients and its obligatory carriers, at the DNA level and in two out of five 3KTD families. This polymorphism was found to be generated by the presence/absence of a TaqI site in intron 9 of the T2 gene. With in vitro amplification of the genomic region around the TaqI site, this RFLP can be detected within 2 days.

Acetyl-CoA C-Acetyltransferase

Interstitial deletion of the long arm of chromosome 11: report of a case and review of the literature.

A 12-month-old female infant with developmental delay, growth retardation, and dysmorphic features including dolichocephaly, telecanthus, ptosis, flat nasal bridge, anteverted nares, high-arched palate, carp-shaped mouth, micro-retrognathia, and low-set and posteriorly rotated ears was found to have an interstitial deletion of chromosome 11 involving bands q14-q22. Immunoblot analysis of her fibroblasts revealed a normal amount of mitochondrial acetoacetyl-coenzyme A thiolase, of which gene locus has been assigned to chromosome 11q22.3-q23.1. This result suggested that the region around the boundary of 11q22.3-q23.1 was intact in this patient.

Abnormalities, Multiple

Analysis of stress-strain curves at fast and slow velocities of loading in vitro in the transverse section of the rat incisor periodontal ligament following the administration of beta-aminopropionitrile.

The in vitro mechanical properties of this ligament were examined by analysing the stress-strain curve obtained from a transverse section of the mandible. Mechanical measures were compared between normal rats and lathyritic rats given drinking water containing 0.2% of beta-aminopropionitrile (BAPN) for 20 days, and between the velocity of loading at 10(4) and 1 mm/24 h. The daily dose of BAPN decreased gradually because the body weight increased gradually. At the velocity of 10(4) mm/24 h, the maximum shear stress, elastic stiffness and failure strain energy density in the experimental subgroup fell to 43-50% of the control values, and at 1 mm/24 h to 71-80%. The maximum strains were not significantly different between the control and experimental subgroups either at 10(4) or at 1 mm/24 h. In the control subgroups, the maximum shear stress, elastic stiffness and failure strain energy density at 1 mm/24 h fell to 0.04-0.30% of those at 10(4) mm/24 h, and in the experimental subgroups to 0.08-0.43%. The maximum strains at 1 mm/24 h were 1.7-1.8 times greater than those at 10(4) mm/24 h in both the control and experimental subgroups. It is assumed that changes in the mechanical properties of the periodontal ligament were caused by inhibition of maturation of the periodontal collagen fibres. Assuming that the periodontal ligament is viscoelastic in nature, it is suggested that the main component reacting at 10(4) mm/24 h was an elastic one and that both components, with emphasis on the viscous one, interact at 1 mm/24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral

Occurrence of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus).

The shore crab (Hemigrapsus sanguineus) is highly resistant to tetrodotoxin (TTX) although it contains no detectable amount of TTX (less than 5 MU/g, where 1 MU is defined as the amount of TTX killing a 20 g mouse in 30 min). Its body fluid was examined for neutralizing effects against the lethal activity of TTX. When the mixture of the body fluid and TTX was injected i.p. into mice, the lethal activity of TTX was significantly reduced; 1 ml of the body fluid was evaluated to neutralize 3.6-4.0 MU of TTX. Higher neutralizing activity (7.2-12.5 MU/ml of the body fluid) was exhibited by i.v. administration of the body fluid into mice before or after i.p. challenge of TTX. The lethal effect of paralytic shellfish poisons was not counteracted by the body fluid. Analysis by gel filtration on Sepharose 6B revealed that the body fluid contained TTX-binding high mol. wt substances (> 2,000,000) responsible for the neutralizing activity of the body fluid against TTX, which accounts for the high resistibility of the crab to TTX. When the crude toxin extracted from the liver of puffer (Takifugu niphobles) was mixed with the body fluid and chromatographed on Sepharose 6B, almost pure TTX was obtained from the fractions containing the TTX-binding high mol. wt substances, suggesting that the TTX-binding high mol. wt substances could be useful in purification of TTX from biological samples.

Animals

Extramedullary plasmacytoma (EMP) of urinary bladder.

An extramedullary plasmacytoma (EMP) is presented with an isolated lesion in the urinary bladder accompanying an IgG-K paraproteinemia. After a short-term oral melphalan administration, the tumor soon regressed together with the paraprotein, and has never recurred during the two-year follow-up. This is the fourth case of primary EMP of the urinary bladder reported in the literature.

Aged

Vitamin B12 improves cognitive disturbance in rodents fed a choline-deficient diet.

The effect of vitamin B12 on learning disturbance was tested in rats. Rats were fed a choline-enriched, choline-deficient, and choline-deficient diet with vitamin B12. Concentrations of acetylcholine in the brain were significantly lower in rats fed a choline-deficient diet than rats fed a choline-enriched diet. Passive avoidance learning shows that rats on a choline-deficient diet showed significantly impaired learning compared to rats on a choline-enriched diet. However, there was no significant difference of acetylcholine in the brain or in the passive avoidance learning between rats fed a choline-enriched and a choline-deficient with vitamin B12 diet. We, therefore, suggest that vitamin B12 potentiates learning in an acetylcholine-deprived brain.

Acetylcholine

Embryonic heart rate of the domestic fowl (Gallus domesticus) in a quasiequilibrium state of altered ambient temperatures.

1. The heart rates (fH) of 12-day-old embryos (young), 16- and 18-day-old embryos (late) and of 20-day-old embryos (externally pipped eggs, EP) were measured noninvasively at a temperature of 38 degrees C and after a 5 hr exposure to an ambient temperature (Ta) within the range 34-46 degrees C. 2. All embryos survived the 5 hr exposure to Ta of 42 degrees C. The lethal Ta ranged from 44 degrees C (EP eggs) to 46 degrees C (young embryos). 3. The temperature coefficient (Q10) of fH became smaller than 2 in EP eggs as Ta was decreased or increased from 38 degrees C, implying an incipient homeothermic response of fH.

Animals

Effects of anticonvulsant drugs on 4-aminopyridine-induced seizures in mice.

The K+ channel blocker 4-aminopyridine (4-AP) causes epileptiform activity in in vitro preparations and is a potent convulsant in animals and man. In mice, 4-AP produces behavioral activation, clonic limb movements and wild running, followed by tonic hindlimb extension and death (ED97, 13.3 mg/kg, s.c.). We evaluated the ability of a series of anticonvulsant drugs to protect against 4-AP-induced seizures using lethality as the endpoint. Drugs with a phenytoin-like profile of activity were protective with ED50 values (all in mg/kg, i.p.) of 34.4 for phenytoin, 18.6 for carbamazepine, 26.9 for felbamate, and 41.5 for zonisamide. Phenobarbital and valproate also protected against 4-AP-induced seizures and lethality (ED50s, 30.6 and 301, respectively). In contrast the NMDA antagonists (+/-)-CPP and (+)-MK-801 were inactive as were the GABA enhancers diazepam, vigabatrin and tiagabine; the antiabsence drug ethosuximide; and the L-type Ca2+ channel blocker nimodipine. We conclude that drugs like phenytoin which block seizure spread are effective antagonists of seizures induced by K+ channel blockade. Drugs with specific actions on other cellular targets may be weak or inactive, presumably because they are unable to attenuate the spread of intense (non-NMDA receptor mediated) excitation evoked by 4-AP.

4-Aminopyridine