Indian childhood cirrhosis-like disease in a Japanese boy undergoing liver transplantation.
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Blood Coagulation↗
Biomedical subjects
A Ohtake
Publications and source records attributed to A Ohtake.
New exfoliative toxin produced by a plasmid-carrying strain of Staphylococcus hyicus.
A new serotype of Staphylococcus hyicus exfoliative toxin (SHET), serotype B, was isolated from the culture filtrate of a plasmid-carrying strain of S. hyicus. The new SHET was purified by precipitation with 70% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column, column chromatography on DEAE-Cellulofine A-500, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The new SHET caused exfoliation of the epidermis as determined by the so-called Nikolsky sign when inoculated into 1-day-old chickens. The new SHET was serologically different from Staphylococcus aureus exfoliative toxins (ETs) (ETA, ETB, and ETC) and from the SHET from the plasmidless strain but showed the same molecular weight as the other serotypes of toxins on SDS-PAGE. It was thermolabile and lost its toxicity after being heated at 60 degrees C for 30 min. We propose that the new SHET be designated SHETB and that the SHET produced by the plasmidless strain be designated SHETA.
Animals↗
Expression analysis of two mutant human ornithine transcarbamylases in COS-7 cells.
Ornithine transcarbamylase (OTC) is located in the mitochondrial matrix of the liver and small intestine and catalyzes the second step of the urea cycle. OTC deficiency (OTCD) is an X-linked inborn error of metabolism and causes hyperammonemia. We reported in 1992 the A152V and G195R mutations in patients with OTCD. These mutant OTC cDNAs were prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The wild-type and mutant cDNAs were transiently expressed in COS-7 cells. The wild-type cDNA gave an OTC activity of 1180 +/- 47 nmol/min per mg protein. The OTC activities of the A152V and the G195R mutants were 3.7% and 2.5% of that of wild-type, respectively. Immunoblot analysis showed that the quantities of OTC proteins in the A152V and G195R mutants were 29% and 12% of that of wild-type, respectively. In pulse-labeling and pulse-chase experiments, the precursor form of OTC was synthesized and processed to the mature form. The A152V mutant OTC was processed to the mature form as rapidly as the wild-type precursor. However, the processed, mature form of the mutant OTC was rapidly degraded, presumably in the mitochondrial matrix. These results indicate that OTCD with the A152V mutation is due both to rapid degradation of the processed, mature form, and to a lower specific activity of the remaining protein.
Animals↗
Morphological and functional changes in coronary vessel evoked by repeated endothelial injury in pigs.
OBJECTIVE: We examined the morphological changes induced by repeated endothelial denudation in coronary artery (CA), as well as functional changes in the endothelium-dependent and smooth muscle responses to various vasoactive agents during the process of intimal thickening. METHODS: We observed vascular responses in denuded and non-denuded portions of pig CA while being fed a normal diet (n = 11, N group) or 2% cholesterol diet (n = 25, C group) to intracoronary acetylcholine (ACh), 5-hydroxytryptamine (5-HT), substance P (SP), and isosorbide dinitrate (ISDN) with and without the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg i.v.) over a period of 8 weeks. Balloon endothelial denudation of the left anterior descending CA was carried out every 2 weeks. RESULTS: In N group, maximum vasoconstriction was obtained with ACh 2 weeks after the first denudation [26 +/- 5% vs. 1 +/- 1% pre-denudation, p < 0.05]. L-NAME did not affect ACh-induced CA diameter changes. Thereafter, the response to ACh was attenuated by repeated denudation in N groups. However, the degree of 5-HT-induced CA narrowing at the denuded portion increased from 7 +/- 4% (0 week) to 88 +/- 8% (8 weeks) (p < 0.05). The changes resulted in severe myocardial ischaemia, and suggested that endothelium-dependent vasodilation was progressively attenuated while hyperreactivity of vascular smooth muscle simultaneously increased. Vasodilation induced by SP was attenuated somewhat, but ISDN-induced vasodilation was preserved. Although mild hypercholesterolaemia was induced in C group, the vascular responses to these vasoactive agents did not differ from those of N group. CONCLUSIONS: Repeated CA endothelial injury and regeneration induce the change of morphology and vascular reactivity in the denuded portion regardless of atherogenic diet. This study strongly suggests that intimal thickening caused by repeated endothelial injury and regeneration induces specific vascular responses to vasoactive agents. Moreover, it is also suggested that during the progression of intimal thickening, increased vascular smooth muscle contraction and decreased endothelium-dependent dilation appear in a stimulus-dependent manner, often leading to severe coronary vasoconstriction accompanied with definitive ECG ST change.
Acetylcholine↗
Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases.
cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.
Animals↗
Binding of chimeric analogs of omega-conotoxin MVIIA and MVIIC to the N- and P/Q-type calcium channels.
Despite their high sequence homology, the peptide neurotoxins omega-conotoxin MVIIA and MVIIC selectively block N- and P/Q-type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by exchanging their N- and C-terminal halves. Binding assay for both N- and P/Q-type calcium channels showed that amino acid residues restricted to the N-terminal half are important for the recognition of N-type channels, whereas essential residues for P/Q-type channel recognition are widely spread over the whole omega-conotoxin molecule.
Amino Acid Sequence↗
Imaging of cAMP-dependent protein kinase activity in living neural cells using a novel fluorescent substrate.
In order to visualize the activity of the cAMP-dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell-permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108-15 cells, PKA activity was localized to the cytosol around the nucleus. In cultured hippocampal neurons, addition of L-glutamate caused PKA activation associated with increase of the cellular cAMP.
Amino Acid Sequence↗
Circular dichroism spectra of calcium channel antagonist omega-conotoxins.
The circular dichroism (CD) spectrum of omega-conotoxin GVIA is quite different from those of omega-conotoxin MVIIA and MVIIC, despite their distinct similarity in three dimensional structures. In order to characterize the unique CD spectrum of omega-conotoxin GVIA, we focused our attention on the aromatic chromophore and analyzed the CD spectra of three synthetic analogs, in which Tyr13, Tyr22, and Tyr27 were individually replaced by alanine. Replacement of Tyr27 caused a significant change in both the near- and far-ultraviolet CD spectrum of omega-conotoxin GVIA and resulted in the omega-conotoxin MVIIA/MVIIC-like pattern, suggesting that Tyr27 has a dominant contribution to the unique CD profile of omega-conotoxin GVIA.
Alanine↗
Amphiphilic helix is essential for the activity of brain injury-derived neurotrophic peptide (BINP).
To study the structure-activity relationships of brain injury-derived neurotrophic peptide (BINP), 12 analogs were synthesized by replacing each amino acid residue with Gly. BINP showed CD spectra typical of an alpha-helical conformation in TFE solution which mimics the membrane environment. In the alpha-helical conformation, BINP showed an amphiphilic profile. Neurotrophic activities of BINP and its analogs were estimated from the effects on supporting septal cholinergic neurons and on rescuing hippocampal neurons from injury caused by glutamate. Both assays showed that the residues on the hydrophobic side of the amphiphilic helix were essential for the neurotrophic activity.
Amino Acid Sequence↗
Imaging of Ca2+/calmodulin-dependent protein kinase II activity in hippocampal neurones.
Our aim was to visualize the dynamic features of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. In order to do so, we synthesized a new reagent by conjugating a fluoroprobe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), to syntide 2, a specific peptide substrate for CaMKII. In cell-free conditions, the conjugate was found to be an effective indicator of calmodulin activation by Ca2+ and the subsequent activation of CaMKII. The reagent is cell-permeable and can stain living cells when bath-applied. Using this technique we were able to obtain fluorescence images of stained cells and analyse the dynamic features of CaMKII inside the cells by means of image processing. Regional heterogeneity of CaMKII activation in cultured hippocampal neurones was seen following L-glutamate administration.
Animals↗
Geometry and lattice formation of surface layers of Sn growing on InSb{111}A,B.
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Journal Article↗
Activation of protein-tyrosine phosphatase SH-PTP2 by a tyrosine-based activation motif of a novel brain molecule.
BIT (a brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (tyrosine-based activation motifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in lambdagt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.
Amino Acid Sequence↗
Two novel gene mutations (Glu174-->Lys, Phe383-->Tyr) causing the "hepatic" form of carnitine palmitoyltransferase II deficiency.
Carnitine palmitoyltransferase II (CPT II) deficiency has two different clinical forms, one with "hepatic" and the other with "muscular" symptoms. We studied the molecular basis of the "hepatic" form in two Japanese siblings. Their CPT II activity in lymphoblasts was reduced to 3% of the level observed in normal controls. cDNA analysis showed that the proband was a compound heterozygote. One allele carried a new mutation, G621-->A (Glu174-->Lys). The other carried three single-base substitutions; a new mutation, T1249-->A (Phe383-->Tyr), and two previously reported polymorphisms. The brother had the same four substitutions. Neither of the two new mutations in this study was detected in the 60 alleles of 30 Japanese control subjects. Secondary structure prediction analysis of the mutated CPT II protein was different from that of the normal protein. We concluded that these mutations caused the "hepatic" form of CPT II deficiency in the probands.
Alleles↗
Molecular analysis of holocarboxylase synthetase deficiency: a missense mutation and a single base deletion are predominant in Japanese patients.
Holocarboxylase synthetase (HCS) deficiency is an inherited disease of biotin metabolism characterized by a unique pattern of organic aciduria, metabolic acidosis, and skin lesions. By analysis of five patients in four unrelated families, two mutations were identified: a transition from T to C which causes an amino-acid substitution of proline for leucine at position 237 (L237P) and a single deletion of guanine (delG1067) followed by premature termination. One patient was homozygous for the L237P mutation, three patients in two families were compound heterozygotes of the missense and deletion alleles, and the other patient was heterozygous for the L237P mutation. Inheritance was successfully demonstrated in all of the patients' families by a modified PCR followed by restriction enzyme digestion. The two mutations accounted for seven of eight mutant alleles, while neither mutation was detected in 108 normal healthy Japanese children (216 alleles). Transient expression in cultured fibroblasts from a patient showed that the L237P mutation was responsible for decreased HCS activity. These results suggest that the L237P and delG1067 mutations are frequent disease-causing mutations in Japanese patients with HCS deficiency. This PCR-based technique may therefore be useful for detecting mutations among Japanese patients.
Amino Acid Sequence↗
A novel tau-tubulin kinase from bovine brain.
During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, beta-tubulin, MAP2 and alpha-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.
Adenosine Triphosphate↗
Tyr13 is essential for the binding of omega-conotoxin MVIIC to the P/Q-type calcium channel.
An analog of omega-conotoxin MVIIC (Y13A-MVIIC) was synthesized by replacing Tyr13 with Ala to study the role of Tyr13 residue conserved in many omega-conotoxins. Y13A-MVIIC has an overall conformation similar to that of the native toxin, but an enormously reduced ability to displace 125I-omega-conotoxin MVIIC binding to rat cerebellar P2 membranes. These results suggest that Tyr13 is essential for the activity of omega-conotoxins at P/Q-type calcium channels.
Amino Acid Sequence↗
Tyr13 is essential for the activity of omega-conotoxin MVIIA and GVIA, specific N-type calcium channel blockers.
Two analogs of omega-conotoxin MVIIA, a 25mer peptide neurotoxin, were synthesized by replacing Lys2 or Tyr13 with Ala. The activities of synthetic analogs were estimated from the inhibitory action on 125I-omega-conotoxin GVIA binding to chick brain synaptic plasma membranes. As in the case of omega-conotoxin GVIA, replacement of Tyr13 resulted in an enormous reduction in activity. In contrast, substitution of Ala for Lys2 gave only a small effect. These results indicate that Tyr13 is a critical amino acid of omega-conotoxin MVIIA and GVIA for blocking N-type calcium channel function.
Amino Acid Sequence↗
Surface phase transition and interface interaction in the alpha -Sn/InSb{111} system.
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Journal Article↗