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H Sakai

Publications and source records attributed to H Sakai.

At least 631 records · Page 35Linked to original sources
PubMed1994

Carbohydrate-mediated recognition of a circulating placental alkaline phosphatase-immunoglobulin M complex.

We detected an abnormal alkaline phosphatase (AP) electrophoretically in the serum of a patient with rheumatoid arthritis, who had a macromolecular AP linked with immunoglobulin M (IgM) bearing a kappa light chain. The IgM isolated from the AP-IgM complex in the patient's serum reacted apparently with all of the AP isozymes tested, i.e. those originating in the liver, bone, intestine and placenta, but the alpha-mannosidase-treated IgM from the patient's serum bound to placental AP (PAP) alone. This suggests that untreated IgM recognizes multivalent epitopes of the AP and that the complex of AP with alpha-mannosidase-treated IgM is a specific antibody-antigen complex. In order to investigate further the multivalent binding capacity for the PAP-untreated IgM complex, we prepared a monoclonal antibody (MoAb) against PAP and identified it as an IgM with a kappa light chain. The binding affinities and their circulating half-lives of the synthetic complexes of PAP and respective MoAbs were examined with and without treatment with several glycosidases. The untreated MoAb bearing IgM had binding affinity for all of the AP isozymes tested, while alpha-mannosidase-treated IgM attached only to PAP, the same as the IgM isolated from the PAP-IgM complex in the patient's serum. The circulating clearance of the PAP-IgM complex in rabbits was faster than either component alone. In addition, the PAP-IgM complex treated with alpha-mannosidase was found to have the shortest half-life of all the complexes of PAP and Igs treated with the several glycosidases tested. These results suggest that the formation of the PAP-IgM complex as an enzyme-linked antibody and the clearance of the complex in vivo are dependent on the sugar moieties of the Igs.

Adult↗
PubMed1994

A GTP-binding protein inhibits a gastric housekeeping chloride channel via intracellular production of superoxide.

A housekeeping basolateral Cl- channel of rabbit gastric parietal cells, the single channel conductance of which is about 0.3 picosiemens, is opened by prostaglandin E2 and closed by intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). In the present patch clamp study, we found a novel GTP gamma S-dependent regulatory mechanism of the Cl- channel. GTP gamma S significantly decreased the open probability of the single Cl- channel without altering unit conductance. An intracellular application of superoxide dismutase (SOD; 100 units/ml) inhibited the GTP gamma S (50 microM)-induced closure of the Cl- channel. SOD plus catalase (100 units/ml) also inhibited the GTP gamma S-induced effect, while catalase alone did not inhibit it. In the absence of GTP gamma S, an intracellular application of hydrogen peroxide (H2O2; 30 microM) did not affect the Cl- channel current. Desferrioxamine (50 microM) which inhibits hydroxyl radical (.OH) production was without effect on the GTP gamma S-induced closure. These results suggest that the GTP gamma S-induced closure of the Cl- channel was due to intracellular production of superoxide (O2.-), but not due to .OH or H2O2. Furthermore, an artificial production of O2.- inside the cell by lumazine (50-100 microM) plus xanthine oxidase (0.5-1 milliunit/ml) in the absence of GTP gamma S also closed the channel. The lumazine/xanthine oxidase-induced closure of the channel was inhibited by SOD, but not by catalase or desferrioxamine. We conclude from these results that GTP-binding protein-coupled production of O2.- leads to closure of the Cl- channel in rabbit gastric parietal cells.

Animals↗
PubMed1994

Purification of a low molecular weight microtubule binding protein from sea urchin eggs.

A low molecular weight microtubule binding protein(SU-MAP34) was purified from sea urchin eggs. This protein bound strongly to the microtubule formed from purified echinoderm tubulin but showed no cross-linking of microtubules. Monospecific antibody against SU-MAP34 was produced and an immunoblotting analysis showed that this protein was not a breakdown product of a protein of a higher molecular mass. Whole cell staining and confocal laser scanning microscope observation showed that SU-MAP34 localized on the filamentous structure of mitotic apparatus and this structure was identified as the microtubule with double staining using anti-SU-MAP34 and anti-tubulin. An immunoblotting experiment showed an enrichment of SU-MAP34 in a microtubule protein fraction prepared using taxol from a crude extract of the cell.

Animals↗
PubMed1994

Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution.

A full-length cDNA clone encoding a purified augmenter of liver regeneration (ALR) factor prepared from the cytosol of weanling rat livers was isolated. The 1.2-kb cDNA included a 299-bp 5' untranslated region, a 375-bp coding region, and a 550-bp 3' untranslated region. It encoded a protein consisting of 125 amino acids. The molecular weight of ALR calculated from the cDNA was 15,081, which is consistent with the size estimated by SDS/PAGE under reducing conditions. The molecular weight of the purified native ALR estimated by SDS/PAGE under nonreducing conditions was approximately 30,000; thus ALR apparently has a homodimeric structure. The recombinant ALR produced by expression of the cDNA in COS cells was tested in vivo in the canine Eck fistula model and found to have potency equivalent to the purified native ALR. The 125-aa sequence deduced from the rat ALR cDNA shows 50% homology to the amino acid sequence of the gene for oxidative phosphorylation and vegetative growth in the yeast Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1994

A protein complex required for signal-sequence-specific sorting and translocation.

We have purified a nascent-polypeptide-associated complex (NAC) which prevents short ribosome-associated nascent polypeptides from inappropriate interactions with proteins in the cytosol. NAC binds nascent-polypeptide domains emerging from ribosomes unless a signal peptide is fully exposed. Depletion of cytosolic proteins (including NAC) from ribosomes carrying nascent polypeptides allows the signal recognition particle (SRP) to crosslink to polypeptides irrespective of whether or not they contain signal peptides. In the absence of cytosol, proteins lacking signal peptides can be mistranslocated into the endoplasmic reticulum in vitro, albeit with low efficiency. Readdition of NAC restores the specificity of SRP and fidelity of translocation.

Amino Acid Sequence↗
PubMed1994

Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130-kDa CryIVA protein produced by Bacillus thuringiensis subsp. israelensis.

There are five amino acid sequences highly conserved among Bacillus thuringiensis delta-endotoxins. We have changed the amino acid residues in block 5, one of the conserved sequences, of CryIVA. When the amino acid residues with charged side chains were replaced by others, the amount of production of the altered CryIVA protein was markedly decreased. It is suggested that the decrease is caused by the unstable conformation of the altered CryIVA protein molecule, as judged by digestion with trypsin and thermolysin. On the other hand, the substitution of amino acid residues in block 5 did not affect the insecticidal activity of CryIVA. These results strongly suggest that block 5 of CryIVA is one of the stability-determining elements of the protoxin molecule.

Amino Acid Sequence↗
PubMed1994

Antiplatelet agents based on cyclooxygenase inhibition without ulcerogenesis. Evaluation and synthesis of 4,5-bis(4-methoxyphenyl)-2-substituted-thiazoles.

The syntheses, biological evaluations, and structure-activity relationships of a series of 4,5-bis-(4-methoxyphenyl)-2-substituted-thiazoles as potent antiplatelet agents with vasodilatory activity are described. 2-Guanidino-4,5-bis(4-methoxyphenyl)thiazole (3), designed from two parent compounds (itazigrel and timegadine), showed inhibitory activity of malondialdehyde (MDA, IC50 = 31 microM) production which is formed from the cyclooxygenase (CO)-catalyzed oxygenation of arachidonic acid in the synthesis of prostanoids in platelets, with vasodilatory activity (ED50 = 2.0 microM). Further structure-activity relationship studies on 3 culminated in the preparation of 4,5-bis(4-methoxyphenyl)-2-[(1-methylpiperazin-4-yl)carbonyl]thiaz ole (10a, FR122047) which exhibited potent inhibitory activity on MDA synthesis in vitro (IC50 = 0.088 microM) and platelet aggregation in guinea pigs ex vivo (100% inhibition even 6 h after 1.0 mg/kg administration) with vasodilatory activity in vitro (ED50 = 6.2 microM). Moreover, 10a demonstrated no ulcerogenesis effect in rats even at 100 mg/kg dosage (safety margin in rats is more than 70 while that of aspirin is only 1.2) in spite of its potent CO inhibition (IC50 = 0.43 microM14), while the use of aspirin, a CO inhibitor and the most popular thromboembolic drug, is restricted by the side effect. Pharmacokinetic studies on 10a have revealed that 10a is detectable in platelet-rich plasma but not in platelet-poor plasma 1 day after oral administration, which indicates that 10a tends to be localized in platelets. This property could be responsible for its low toxicity and reduction of side effects in clinical studies.

Animals↗
PubMed1994

Development of voltage-dependent inward currents in dissociated newt retinal pigment epithelial cells in culture.

The whole-cell patch-clamp technique was employed to investigate the development of voltage-gated currents in cultured newt retinal pigment epithelial (RPE) cells. Freshly dissociated RPE cells were inexcitable and mainly exhibited a voltage-gated outward K+ current. Within a few days in culture RPE cells began to flatten and gradually became less pigmented. During subsequent time in culture the cells proliferated and formed monolayer colonies containing depigmented cells. About 36% cells (15/42) examined between 6 and 21 days in culture developed the voltage-gated inward currents. In two depigmented cells cultured for more than 17 days, the sustained inward Ca2+ current was developed. The result suggests that RPE cells develop the voltage-gated inward currents responsible for retinal neurones with time in culture.

Animals↗
PubMed1994

C-terminal topology of gastric H+,K(+)-ATPase.

An antibody was prepared against a peptide corresponding to residues 1024-1034 (the putative C-terminus) of the alpha-subunit of hog gastric H+,K(+)-ATPase. The antibody bound to a 95 kDa band of H+,K(+)-ATPase that was solubilized in SDS, but not to that of Na+,K(+)-ATPase. It also bound to products of tryptic digestion that included C-terminal fragments of the H+,K(+)-ATPase alpha-subunit. The same amount of the antibody bound to both intact (tight) and lyophilized (leaky) inside-out gastric vesicles, indicating that its epitope is present on the cytosolic side of the vesicles. This finding was further confirmed by using fluorescence-immunolocalization techniques and streptolysin-O to permeabilize newt oxyntic cells. Stimulation of isolated newt oxyntic cells with dibutyryl cyclic AMP induces fusion of tubulovesicles with the apical membrane, so that the luminal domains of the H+,K(+)-ATPase alpha-subunit directly face the cell-suspension medium. The antibody did not bind to the stimulated intact cell, but bound to cells permeabilized with streptolysin-O, indicating that it binds from the cytoplasmic side to the C-terminus of the H+,K(+)-ATPase alpha-subunit in apical and tubulovesicular membrane, and also that the H+,K(+)-ATPase alpha-subunit has an even number of transmembrane domains.

Amino Acid Sequence↗
PubMed1994

Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV). If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.

Bacterial Proteins↗
PubMed1994

Functional roles of Trp337 and Glu632 in Clostridium glucoamylase, as determined by chemical modification, mutagenesis, and the stopped-flow method.

Chemical modification of glucoamylase (EC 3.2.1.3) from Clostridium sp. G0005 (CGA) with N-bromosuccinimide (NBS) was carried out in the presence or absence of an inhibitor, acarbose. CGA lost its catalytic activity through NBS oxidation in the absence of acarbose. The absorbance change at 280 nm suggested that acarbose protects about 2 Trp residues from NBS oxidation. We performed peptide mapping analysis to identify the protected Trp residues, and Trp321, Trp337, Trp433, and Trp569 were identified as candidates to be protected by acarbose. These 4 Trp residues were replaced by site-directed mutagenesis with Phe. The Trp337-->Phe mutant showed very weak catalytic activity, so Trp337 is proposed as an important residue for the catalytic activity. Further, we constructed a Glu632-->Gln mutant. Glu632 is the putative catalytic base. The presteady-state kinetics of the Trp337-->Phe and Glu632-->Gln mutants and the wild-type CGA were investigated using maltotriose as a substrate. The reaction of wild-type CGA can be explained as one involving three intermediates. On the other hand, the two mutants' reactions are explained by a two-step mechanism lacking the third intermediate. Trp337 and Glu632 appear to be crucial for the formation of the third intermediate in the wild-type reaction, which precedes the transition state.

Acarbose↗
PubMed1994

Functional features of oriV of the broad host range plasmid RSF1010 in Pseudomonas aeruginosa.

The broad host range plasmid RSF1010 requires for its replication in Escherichia coli three plasmid-encoded proteins and specific nucleotide sequences ssiA, ssiB, and iterons in the oriVRSF1010. In Pseudomonas aeruginosa, a recombinant mini-RSF1010 plasmid lacking ssiB lost its replication ability, but a miniplasmid lacking ssiA or carrying a primosome assembly site in place of ssiA could replicate. Moreover, ssiA, as a sole ssi signal, in the orientation that ssiB had originally taken was sufficient for replication of the miniplasmid. These results indicated that only one RSF1010-specific ssi signal in the orientation that ssiB takes in wild-type oriVRSF1010 was essential for replication of RSF1010. Replication of the miniplasmids was dependent on the three plasmid-encoded proteins, RepA, B', and C, as in E. coli.

Bacteriophage phi X 174↗
PubMed1994

Immunoelectron microscopic localization of HNK-1 in the embryonic rat heart.

To confirm the role of HNK-1 in conduction tissue, the ultrastructural localization of monoclonal antibody HNK-1 was analyzed in developing rat hearts at embryonal day 14.5 by immunoelectron microscopic labeling procedures with post-embedding immunogold staining. Tissue sections in different planes containing the sino-atrial (SA) node, atrio-ventricular (AV) node and His bundle were used to demonstrate HNK-1. Immunogold labeling was detected on the cell surfaces and in the extracellular matrices of cells that had features common to conduction tissue cells. Non-specialized contractile myocytes were not labeled by this antibody. Furthermore, immunogold labeling was more prominent in wide intracellular spaces than in narrow intercellular spaces, and rarely observed in cell-cell contact regions. The cell surfaces and extracellular matrices of mesenchymal cells in the endocardial cushion, which contacts the His bundle, were also positive, suggesting the involvement of tract formation to the AV node. These findings may indicate that HNK-1 plays an important role in cell-cell adhesion processes both temporally and spatially in the developing conduction tissue. It was concluded, therefore, that HNK-1 is a suitable marker of the embryonic heart conduction system and might be useful in analyzing anomalous conduction systems, as in congenital heart disease.

Animals↗
PubMed1994

Distribution of acetylcholinesterase activity in the rat embryonic heart with reference to HNK-1 immunoreactivity in the conduction tissue.

Acetylcholinesterase (AChE) activity was topographically investigated in the presumptive cardiac conduction tissue regions visualized by HNK-1 immunoreactivity in rat embryos, and AChE-positive cells were examined with the electron microscope. On embryonic day (ED) 14.5, when HNK-1 was most intensely visualized, AChE activity could not be detected enzyme-histochemically in the conduction tissue regions, except in the ventricular trabeculae and part of the AV node. On ED 16.5, however, the AChE activity was clearly demonstrated in some parts of the developing conduction tissue. One exception was the AV node region, where an AChE-positive area was in close proximity to an area showing HNK-1 immunoreactivity but did not overlap. Furthermore, AChE activity was demonstrated predominantly in the ventricular trabeculae, including cardiac myocytes, but was rather weak in the atrium. With the electron microscope, AChE reaction products were observed predominantly intracellularly in both developing conduction tissue cells and developing ordinary myocytes, and no reactivity was found in neuronal components. From ED 18.5 until birth, both AChE activity and HNK-1 immunoreactivity faded away in the conduction tissue. Thus, transient AChE activity in the embryonic heart seems to be different from the developing adult form and may be related to a morphogenetic function in embryonic tissues, as proposed by other authors.

Acetylcholinesterase↗
PubMed1994

Chemo-endocrine therapy for prostate cancer with bone metastasis. Nagasaki Prostate Cancer Research Group.

We analyzed the clinical effects of initial chemoendocrine therapy on 31 prostate cancer patients with bone metastasis. These patients had been newly diagnosed between 1983 and 1991 and had received no previous therapy. As endocrine therapy, the patients received 1 mg ethynylestradiol daily with or without orchiectomy. In addition, they received three courses of chemotherapy consisting of 20 mg/m2 cisplatin given on days 1, 3, and 5 and 20 mg/m2 Adriamycin or 40 mg/m2 epirubicin given on day 5. Subsequently, for maintenance therapy, the patients received 1 mg ethynylestradiol and 150 mg 5-fluorouracil [or 300 mg tegafur plus uracil (UFT)] daily. Patients given our regimen of chemoendocrine therapy had a significantly better prognosis than did the controls treated with endocrine therapy alone (P = 0.05), although treatment was not randomized. The cause-specific survival rates at 5 years for the chemoendocrine-therapy patients and the control group were 65.4% and 37.4%, respectively. A multivariate analysis of possible prognostic factors, i.e., age, histological grade, prostatic acid phosphatase, tumor-related pain, the extent of disease (EOD) on bone scan, and the type of initial treatment, confirmed that the initial treatment (P = 0.03) and the EOD grade (P = 0.05) had a significant effect on survival. On the basis of these results, it is necessary to carry out a randomized trial to compare our chemoendocrine regimen with endocrine therapy alone in untreated patients with advanced prostate cancer.

Aged↗
PubMed1994

Ringed grafting for an abdominal aortic aneurysm in a 92-year-old patient: report of a case.

We report herein the case of a 92-year-old man with an abdominal aortic aneurysm who underwent successful surgical resection using a ringed graft. His postoperative course was uneventful, and he was discharged after 42 days, following which he attended the outpatient department twice a month for regular check-ups. He finally died of pneumonia which developed from an upper respiratory tract infection 6 months after his operation. Thus, surgical treatment should always be considered when an aneurysm is detected, even in very aged patients for whom the activities of daily living are possible.

Aged↗
PubMed1994

Delayed gastric emptying in patients with liver cirrhosis.

Using a scintigraphic technique, we investigated gastric emptying of a semisolid meal in 20 patients with liver cirrhosis and in 10 control subjects and correlated gastric emptying with gastrointestinal symptoms and with autonomic nervous function as determined by R-R interval variation on the electrocardiogram. All subjects lacked endoscopic abnormalities that might explain their gastrointestinal symptoms. None had alcoholic liver disease, diabetes, or other diseases known to affect gastric emptying. The half-time for gastric emptying was significantly prolonged in the cirrhotic patients (51.3 +/- 16.6 minutes) as compared with control subjects (29.9 +/- 8.4) (P < 0.01). There was a significant correlation between the gastrointestinal symptom score and the half-time for gastric emptying (r = 0.46, P < 0.05) in the cirrhotic patients. However, the decreases in R-R interval variation and gastric emptying were not significantly correlated. These observations indicate that delayed gastric emptying is frequently present in patients with liver cirrhosis and may produce their gastrointestinal symptoms.

Autonomic Nervous System↗