Biomedical subjects

M Ishiguro

Publications and source records attributed to M Ishiguro.

At least 73 records · Page 4Linked to original sources

PubMed1998

Complete amino acid sequences of chitinase-1 and -2 from bulbs of genus Tulipa.

The complete amino acid sequences of tulip bulb chitinase-1 and -2 (TBC-1 and -2) were determined. The sequences of the TBC-1 and TBC-2 were solved by analysis of peptides derived by enzymatic digestions as well as by chemical cleavages with cyanogen bromide (CNBr), o-iodosobenzoic acid, and hydroxylamine. TBC-1 and TBC-2 both consisted of 275 amino acid residues and had molecular masses of 30,825 and 30,863, respectively. They shared 247 identical residues, that is 90% identity. Comparison of their sequences with that of gladiolus bulb class IIIb chitinase-a (GBC-a) showed that 63% of the residues of both TBC-1 and TBC-2 are identical to that of GBC-a. From these results, it was seen that TBC-1 and -2 are class IIIb chitinases. The characteristic difference in specific activity between TBC-1 and -2 was also discussed on the basis of their amino acid sequences.

Amino Acid Sequence↗

PubMed1998

Properties of alpha-mannosidase partially purified from the apple snail, Pomacea canaliculata.

Pomacea canaliculata alpha-mannosidase (260 kDa), composed of at least two isoforms with different pI, was partially purified. The activity was maximum at pH 4 and unaltered after incubation at 60 degrees C for 60 min. ZnCl2, CaCl2, NaCl, and SH-reagents increased the activity, while MnCl2 and EDTA inhibited it. The enzyme catalyzed the hydrolysis of alpha 1-2, alpha 1-3, and alpha 1-6 mannosidic linkages.

Animals↗

PubMed1998

Complete amino acid sequence of chitinase-a from bulbs of gladiolus (Gladiolus gandavensis).

The complete amino acid sequence of gladiolus bulb chitinase-a (GBC-a) was determined. First the tryptic peptides from GBC-a after it was reduced and S-carboxymethylated were sequenced and then the peptides were further studied by chemical cleavage of the enzyme. GBC-a consisted of 274 amino acid residues and had a molecular mass of 30,714 Da. Two consensus sequences essential for chitinase activity by plant class III chitinases were conserved in GBC-a, although its sequence similarity with plant class III chitinases was less than 20%. Sequence comparison of GBC-a with sequences of other proteins in a protein identification resource (PIR) showed that the GBC-a sequence was 33% similar to that of narbonin, a seed storage 2S globulin from narbon beans.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of chitinase isoforms from the bulbs of four species of the genus Tulipa.

Six chitinase isoforms, designated TBC-1 to TBC-6, were purified to homogenity from the bulbs of four species (Tulipa bakeri, T. tarda, T. turkestanica, and T. praestans) of the genus Tulipa by CM-cellulose column chromatography, Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S fast protein liquid chromatography (FPLC). The chitinases had molecular weights of 30,000 and isoelectric points of 5.2 to 6.1. These chitinases were found to proteins with similar amino acid compositions and N-terminal sequences. The tulip chitinases all had two half-cystine residues, one more than gladiolus bulb class IIIb chitinase, but many fewer than chitinases of plant class I (15-17 Cys residues/mol), II (5-8 Cys residues/mol), or III (6 Cys residues/mol). The N-terminal sequences of tulip chitinases were similar to the sequence of the gladiolus chitinase, but did not resemble sequence of any class of plant chitinase. The optimal pH of these chitinases toward glycolchitin was pH 5. TBC-1 hydrolyzed (GlcNAc)6 into (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and hydrolyzed (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.

Chitin↗

PubMed1998

Complete amino acid sequence of chitinase-A from leaves of pokeweed (Phytolacca americana).

The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.

Amino Acid Sequence↗

PubMed1998

[Intracranial hemorrhage observed in the cases of cervical internal carotid artery occlusion associated with moyamoya disease-like vessels].

We report on two peculiar cases of intracranial hemorrhage due to the rupture of moyamoya disease-like vessels associated with unilateral internal carotid occlusion at its origin. The first case is 44-year-old male showing intraventricular hemorrhage associated with right internal carotid occlusion. The second case is 58-year-old female presenting sub-arachnoid hemorrhage associated with left internal carotid occlusion. Although both cases showed the unique appearance of collateral flow resembling moyamoya disease, they were not classified as moyamoya disease nor unilateral Moyamoya-like state. Vascular abnormality such as cerebral aneurysm and arteriovenous malformation were not at all detected. The etiology of hemorrhage for both cases is presumed as the rupture of moyamoya disease-like vessels, however, it is unable for us to determine why the one case showed IVH, yet the other showed SAH. The two cases showed hypoperfusion of ipsilateral cerebral hemisphere on SPECT that followed by the external-internal carotid revascularization surgery in the chronic stage. Post operative study proved the improvement of the CBF reserve. Nevertheless, whether the reconstructive vascularization prevent an occurrence of rebleeding needs the longer observation and the accumulation of the resembling cases.

Adult↗

PubMed1997

A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom.

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

Amides↗

PubMed1997

Purification, characterization, and synthesis of three novel toxins from the Chinese scorpion Buthus martensi, which act on K+ channels.

Three novel toxins belonging to the scorpion K+ channel-inhibitor family were purified to homogeneity from the venom of the Chinese scorpion Buthus martensi. They have been identified according to their molecular mass (3800-4300 Da) and their neurotoxicity in mice and characterized as 37-amino acid peptides. One of them shows 81-87% sequence identity with members of the kaliotoxin group (named BmKTX), whereas the other two, named BmTX1 and BmTX2, show 65-70% identity with toxins of the charybdotoxin group. Their chemical synthesis by the Fmoc methodology allowed us to show that BmKTX, unlike BmTX1 and BmTX2, possesses an amidated C-terminal extremity. Toxicity assays in vivo established that they are lethal neurotoxic agents in mice (LD50s of 40-95 ng per mouse). Those toxins proved to be potent inhibitors of the voltage-gated K+ channels, as they were able to compete with [125I]kaliotoxin for its binding to rat brain synaptosomes (IC50s of 0.05-1 nM) and to block the cloned voltage-gated K+ channel Kv1.3 from rat brain, expressed in Xenopus oocytes (IC50s of 0.6-1.6 nM). BmTX1 and BmTX2 were also shown to compete with [125I]charybdotoxin for its binding to the high-conductance Ca2+-activated K+ channels present on bovine aorta sarcolemmal membranes (IC50s of 0.3-0.6 nM). These new sequences show multipoint mutations when compared to the other related scorpion K+ channel toxins and should prove to be useful probes for studying the diverse family of K+ channels.

Amino Acid Sequence↗

PubMed1997

5,6-Cis-penems: broad-spectrum anti-methicillin-resistant Staphylococcus aureus beta-lactam antibiotics.

5,6-cis-Penem derivatives have been synthesized and evaluated as anti-MRSA antibiotics. The cis-penems 5 and 6 showed potent activities against not only MRSA but also a wide variety of bacteria including beta-lactamase-producing microorganisms. These compounds were designed to have high affinity to the penicillin-binding protein 2a of MRSA and to form stable acyl intermediates with beta-lactamases by blocking the deacylating water molecule.

Anti-Bacterial Agents↗

PubMed1997

Structure-activity relationships of penem antibiotics: crystallographic structures and implications for their antimicrobial activities.

Twelve closely related crystal structures of the penem derivatives revealed a characteristic short contact of the oxygen atom in the C2 side-chains with the S1 atom. The side-chain conformations of the crystal structures showed a good correlation with the antimicrobial activity. In particular, the penems which show high antimicrobial activity have similar torsion angles for S1-C2-C1'-C2', suggesting that the disposition of the C2' atom would be important for binding to penicillin-interacting enzymes. Two conformations of the C6 hydroxyethyl group were observed in the crystal structures. Of those two, the conformation with a larger torsion angle (delta = 179.2 degrees) is deduced to be the enzyme-bound conformation in the Michaelis complex.

Anti-Bacterial Agents↗

PubMed1997

Inhibition of fructose-6-phosphate,2-kinase by N-bromoacetylethanolamine phosphate in vitro and in vivo.

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-BPase), a bifunctional enzyme, catalyzes the synthesis and degradation of a potent activator, fructose-2,6-bisphosphate (Fru-2,6-P2), of phosphofructokinase, and has been postulated to be an important enzyme in the regulation of glycolysis in mammalian tissues. The purpose of this study was to determine whether or not N-bromoacetylethanolamine phosphate (BrAcNHEtOP), a specific active site-directed inactivator of Fru-6-P,2-kinase, is useful for studies on the role of Fru-6-P,2-kinase in the regulation of glycolysis in vivo. BrAcNHEtOP inactivated purified recombinant rat testis-type Fru-6-P,2-kinase as well as Fru-6-P,2-kinase in a rat liver extract, with half maximum inactivation concentrations of 2 and 15 mM, respectively, on 30 min incubation at 30 degrees C. The increases in Fru-6-P,2-kinase activity and the Fru-2,6-P2 concentration in livers, prepared from fasted rats, induced by high glucose (50 mM) perfusion were suppressed in parallel after pre-perfusion with 1 to 10 mM BrAcNHEtOP, dose-dependently. Five hours after intraperitoneal injection of BrAcNHEtOP (50 to 150 mg/kg) into mice, the Fru-6-P,2-kinase activity and Fru-2,6-P2 concentration in livers had decreased in parallel, dose-dependently. These effects continued for 24 h and were accompanied by decreases in the fructose-1,6-bisphosphate, triose phosphates, and lactate contents, although the contents of glucose-6-phosphate and fructose-6-phosphate did not change. These results suggested that BrAcNHEtOP inactivates Fru-6-P, 2-kinase, resulting in a decrease in the Fru-2,6-P2 level, which causes inactivation of phosphofructokinase and consequently inhibition of glycolysis in liver. Furthermore, the suppressed levels of Fru-6-P,2-kinase activity and metabolites in mice livers were sustained by daily injection of BrAcNHEtOP for 4 days, and body weight gain was also suppressed during the administration of BrAcNHEtOP. These results suggested that BrAcNHEtOP will be a useful reagent for studying the role of Fru-6-P,2-kinase in vivo.

Animals↗

PubMed1997

Renal primitive neuroectodermal tumor: a morphologic, cytogenetic, and molecular analysis with the establishment of two cultured cell lines.

We report two patients with renal primitive neuroectodermal tumor (PNET) in whom the diagnosis was established by both a cytogenetic and a molecular analysis. Histologically, both renal tumors were composed of uniform immature round cells with a positive immunoreactivity for O13 (p30/32 MIC2). The cytogenetic analysis with in situ hybridization (chromosome painting) demonstrated reciprocal translocation t(11;22)(q24;q12) specific to PNET in the cultured cells derived from each tumor. The reverse transcriptase-polymerase chain reaction (RT-PCR) in both tumors demonstrated EWS/ FLI-1 fusion transcripts, representing the molecular equivalent of t(11;22). A Southern blot analysis also confirmed EWS gene rearrangement in both renal tumors. In addition, the authors also established two new cell lines (designated as FU-RPNT-1 and FU-RPNT-2) from renal PNETs. When transplanted into athymic mice, FU-RPNT-1 and FU-RPNT-2 reproduced and maintained the morphologic and molecular characteristics of the original tumors. In conclusion, the detection of t(11;22) and EWS/FLI-1 fusion transcripts is considered to provide a novel adjunctive method for diagnosing renal PNET. These newly established cell lines thus may be used to investigate the biologic behavior related to renal PNETs.

Adult↗

PubMed1997

GB virus C and hepatitis C virus infections in hemodialysis patients in eight Japanese centers.

RNA of a putative non-A to E hepatitis virus, designated GB virus C (GBV-C), was detected in 40 (6.2%) of 645 hemodialysis patients, at a frequency significantly higher than in 3 (0.9%) of 336 blood donors in Japan (p < 0.001). A history of transfusion was more frequent (88 vs. 58%, p < 0.001), the duration of dialysis was longer (13.2 +/- 7.9 vs. 7.9 +/- 6.5 years, p < 0.001), and the detection of hepatitis C virus RNA was more often (38 vs. 18%, p < 0.01) in the 40 patients with GBV-C RNA than in the 605 patients without it. The prevalence of GBV-C RNA varied widely from 0 to 10% among the 8 dialysis centers. These results indicate that hemodialysis patients would be at increased risk of GBV-C transmitted by transfusions. The detection of GBV-C RNA in the 5 patients without a history of transfusion and a high prevalence restricted to certain dialysis centers would reflect nosocomial infection.

Alanine Transaminase↗

PubMed1997

Neurotropic pyrimidine heterocyclic compounds. II. Effects of novel neurotropic pyrimidine derivatives on astrocytic morphological differentiation.

Astrocytes play an important role in supporting nerve regeneration after brain injuries. In this study, the effects of novel neurotropic synthesized pyrimidine compounds on astrocytic morphological differentiation were examined. Treatment of protoplasmic cultured astrocytes with 2-piperidino-5,6-dihydro-7-methyl-6-oxo(7 H)pyrrolo[2,3-d]pyrimidine maleate (MS-430) and the related compounds caused astrocytic process formation in 60 min. The morphology of MS-430-treated cells was similar to that of dibutyryl cAMP (DBcAMP)-treated cells. The astrocytic process formation by MS-430 was observed within 60 min and the maximum effect was obtained at the drug concentration of 0.5-1.0 mM. Rhodamine-phalloidin staining showed that astrocytic cytoskeletal actin was reorganized by MS-430 and DBcAMP. MS-430 did not increase cAMP accumulation in cultured astrocytes. These results suggest that the neurotropic pyrimidines induced astrocytic morphological differentiation through a cAMP-independent mechanism.

Animals↗

PubMed1997

Further studies on thermal denaturation of pyruvate dehydrogenase complex from Bacillus stearothermophilus.

Thermally induced changes in pyruvate dehydrogenase complex (PDC) from B. stearothermophilus were examined mainly at temperatures from 60 degrees to 70 degrees C. Accompanied by inactivation of pyruvate decarboxylase, light scattering decreased, and ANS fluorescence increased. These changes including the inactivation were approximately first-order reactions, and the values of rate constants were greatly dependent on temperature. Chromatographic studies showed that any polypeptides were in associated forms and that final products were aggregates (> 230S) and an assembly (48S) smaller than PDC. The aggregates and assembly were rich in decarboxylase and lipoate acetyltransferase, respectively. It was suggested that, during the thermal denaturation, a decarboxylase was dissociated from PDC and immediately involved in aggregates.

Chromatography, Gel↗

PubMed1997

Purification and characterization of two chitinase isoforms from the bulbs of gladiolus (Gladiolus gandavensis).

Two chitinase isoforms, designated GBC-a and GBC-b, were purified from the bulbs of gladiolus (Gladiolus gandavensis) using CM-cellulose column chromatography followed by Butyl-Toyopearl 650 M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S FPLC. GBC-a and GBC-b are weakly acidic and weakly basic proteins with molecular masses of 30 kDa, and isoelectric points of 6.0 and 7.5, respectively. GBC-a and GBC-b were found to be homologous proteins with similar amino acid compositions and N-terminal sequences. The number of half-cystine residues in GBC-a and GBC-b was only one each, which is much lower than those of plant class I (15-17 Cys residues/mol), class II (5-8 Cys residues/mol), and class III (6 Cys residues/mol) chitinases. The N-terminal sequences of GBC-a and GBC-b were completely different from those of plant three classes of chitinases. The optimal pHs of these chitinases toward glycolchitin were pH 5. GBC-a hydrolyzed (GlcNAc)5 into (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4, and (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.

Amino Acid Sequence↗

PubMed1997

The amino acid sequence of mitogenic lectin-B from the roots of pokeweed (Phytolacca americana).

The complete amino acid sequence of pokeweed lectin-B (PL-B) has been analyzed by first sequencing seven lysylendopeptidase peptides derived from the reduced and S-pyridylethylated PL-B and then connecting them by analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides linked to Asn96 and Asn139, and has a molecular mass of 34,493 Da. PL-B is composed of seven repetitive chitin-binding domains having 48-79% sequence homology with each other. Twelve amino acid residues including eight cysteine residues in these domains are absolutely conserved in all other chitin-binding domains of plant lectins and class I chitinases. Also, it was strongly suggested that the extremely high hemagglutinating and mitogenic activities of PL-B may be ascribed to its seven-domain structure.

Amino Acid Sequence↗

PubMed1997

Developmental changes in functional EEG asymmetry: a multivariate autoregressive modeling approach.

To investigate the functional asymmetry of the electroencephalogram (EEG) in the developing brain, we analyzed quantitatively the different components of simulated EEG (SEEG) in a model before and after cutting off the intercerebral interaction. Original EEG (OEEG) recordings were obtained from 374 normal children aged 3-12 years. The SEEGs were computed from the OEEG after applying 'ARdock', a new multivariate autoregressive (AR) modeling method. Subsequently, the univariate AR and component analysis was applied on OEEG and SEEG. The F value was computed from Mahalanobis' distance between both AR coefficients of component activities for age, EEG derivation and frequency band. Our analysis showed developmental lateralities in the alpha band at the frontal pole in 5 and 12 year old children, and at the central region in 8 and 11 year old children. Our results suggest that the intercerebral interactions develop at approximately 5 years of age at the left frontal pole during the period of maturation of alpha band activity and after 8 years of age when it replaced theta band activity. The present method of analysis may have a wide clinical application in the analysis of normal and abnormal EEGs.

Brain↗