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Biomedical subjects

H Sakai

Publications and source records attributed to H Sakai.

At least 559 records · Page 31Linked to original sources
PubMed1995

A gastric housekeeping Cl- channel activated via prostaglandin EP3 receptor-mediated Ca2+/nitric oxide/cGMP pathway.

Prostaglandin E2 (PGE2) has a cytoprotective role in the gastric parietal cell. PGE2 opened a housekeeping basolateral Cl- channel of rabbit gastric parietal cells, the single channel conductance of which was about 0.3 picosiemens. In the present patch-clamp and Fura 2 fluorescence studies, we found that PGE2 increased the intracellular free Ca2+ concentration ([Ca2+]i) and that PGE2-induced opening of the Cl- channel depended on the increase of [Ca2+]i. A novel bifunctional prostaglandin EP3 agonist/EP1 antagonist, 5(Z)-7-[1S, 2S, 3S, 5R)-3-(trans-beta-styren) sulfonamido-6,6-dimethylbicyclo- (3.1.1)hept-2-yl]-5-heptenoic acid, also increased both [Ca2+]i and channel opening. The PGE2-induced effect was mediated via production of nitric oxide (NO); that is, NG-monomethyl-L-arginine, an inhibitor of NO production, markedly inhibited the PGE2-induced channel opening, and nitroprusside, a NO donor, induced the channel opening in the absence of PGE2. Both PGE2 and A23187, Ca2+ ionophore, elevated the cGMP content of isolated parietal cells. The A23187-induced channel opening was abolished by methylene blue, a guanylate cyclase inhibitor. In conclusion, we found that the PGE2-induced opening of the housekeeping Cl- channel in the parietal cell involves the EP3 receptor-mediated increase in [Ca2+]i via a pertussis toxin-sensitive GTP-binding protein, resulting in successive production of NO and cGMP.

Animals↗
PubMed1995

Deletion and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: effects on its activity.

In order to elucidate a role of a putative EF-hand motif (144-172) in phospholipase C-delta 1 (PLC-delta 1), deletion and point mutation of the enzyme were performed and the mutated cDNAs were expressed in CHO cells and E. coli AD202 strain. Deletion of amino acid residues of 141-236 or 173-236 resulted in abolition of PLC activity. However, the decreased PLC activity to 15-20% by deletion of the EF-hand motif (144-172) was still Ca(2+)-dependent. Furthermore, mutants, in which conserved Asp153, Asp157, Glu164 or all these acidic amino acids in the EF-hand motif were replaced with alanine residues, showed nearly the same PLC activity and Ca(2+)-dependency as those of wild-type. These results suggest that the region containing the EF-hand motif may not play a role in regulation of Ca(2+)-sensitivity of PLC-delta 1, but is important for its activity.

Alanine↗
PubMed1995

Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum.

We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding.

Animals↗
PubMed1995

Rev-dependency of expression of human immunodeficiency virus type 1 gag and env genes.

Structural gene expression of human immunodeficiency virus type 1 (HIV-1) requires a viral regulatory protein, Rev transactivator. We investigated Rev-dependency of HIV-1 gene expression by various reporter systems. Expression of unspliced and single-spliced viral mRNAs was demonstrated to be differentially dependent on the Rev function. This difference of Rev-dependency was found not to be determined by cis-elements in gag, pol, and env coding sequences reported so far, and was lost when the reporter constructs containing minimum elements for Rev-responsiveness such as splice signals and rev responsive element were used for experiments. These findings indicated that the fundamental structure of HIV-1 mRNA was critical for the differential regulation of gene expression by Rev transactivator.

Animals↗
PubMed1995

Expression of the genes for insecticidal crystal proteins in Bacillus thuringiensis: cryIVA, not cryIVB, is transcribed by RNA polymerase containing sigma H and that containing sigma E.

To investigate the mechanism of transcriptional regulation of cryIVA and cryIVB, encoding 130-kDa dipteran-active crystal proteins, in Bacillus thuringiensis subsp. israelensis, we introduced each gene into several sporulation mutants of Bacillus subtilis. A spoIIG mutation, the wild-type gene of which encodes sigma E precursor, completely blocked the cryIVB transcription. In contrast, low but detectable transcription of cryIVA was observed in the spoIIG mutant. In the wild-type B. subtilis, no transcription of cryIVB was detected before T2 (2 h after the onset of stationary phase), while the cryIVA transcription started at the late exponential phase at low levels. Furthermore, in a wild-type strain of B. thuringiensis subsp. israelensis, transcription of cryIVA began earlier than that of genes encoding other crystal components, cryIVB and cytA. A consensus sequence recognized by an RNA polymerase containing sigma H of B. subtilis was found upstream of the transcription start point of cryIVA, which overlapped with that recognized by sigma E.

Bacillus subtilis↗
PubMed1995

Cloning and characterization of a Bacillus thuringiensis homolog of the spoIIID gene from Bacillus subtilis.

The SpoIIID protein of Bacillus subtilis (Bs) is a small DNA-binding protein that is essential for gene expression of the mother cell compartment during sporulation. We have cloned a DNA fragment from Bacillus thuringiensis (Bt) that showed a specific hybridization with the Bs spoIIID gene. Sequence analysis found an open reading frame encoding 90 amino acids (aa), which are 89% identical to the deduced aa sequence of Bs spoIIID. Upstream from the transcription start point (tsp), a nucleotide sequence highly homologous to the consensus sequence motif for the sigma 35-recognized promoters was found. Northern blot analysis has indicated that the expression of the gene is induced only at the midsporulation stage, and that the gene constitutes an operon with a downstream gene, mreB. The Bs strain carrying the spoIIID delta erm or spoIIID83 mutation completely restored sporulation ability upon introduction of the spoIIID homologous gene from Bt. These results strongly suggest that the gene we have cloned is a Bt homolog of spoIIID.

Amino Acid Sequence↗
PubMed1995

Transjugular intrahepatic portosystemic shunt for Budd-Chiari syndrome after failure of surgical shunting.

A case of acute Budd-Chiari syndrome in a 26-year-old woman is reported. After a mesocaval shunt, the patient remained asymptomatic for 21 months, but ascites and hepatomegaly reappeared due to inferior vena cava stenosis subsequently treated by balloon dilation. Recurrence of stenosis indicated the need for a cavoatrial shunt with an expanded polytetrafluoroethylene prosthesis, which was followed by a complete recovery during the next 29 months. Radiological follow-up with magnetic resonance imaging demonstrated progressive hepatomegaly, thrombosis of the cavoatrial shunt, and stenosis of the mesocaval shunt. A transjugular intrahepatic portosystemic shunt was carried out, despite the absence of any patent residual hepatic vein at the usual level, by perforating the inferior vena cava and liver up to the right portal vein. An expandable 12-mm stent was successful in decreasing liver congestion. Dilation of the transjugular intrahepatic portosystemic shunt was done 15 months later, and the patient remains asymptomatic after a follow-up of 18 months.

Adult↗
PubMed1995

Chemo-endocrine therapy in patients with stage D2 prostate cancer.

There have only been a few studies of chemo-endocrine therapy compared with endocrine therapy alone in newly diagnosed prostate cancer patients. We assessed the effects of these two therapies by comparing long-term survival rates. One hundred and twenty-nine patients were entered in this study between November 1977 and March 1992. Seventy-seven patients were treated with endocrine therapy alone. Other 52 patients received chemo-endocrine therapy, which included orchiectomy and/or diethylstilbestrol diphosphate (DES-DP) plus Cisplatin, with or without other cytotoxic agents. All patients had bone metastasis at the beginning of the study. There was a significant difference in survival between patients who received endocrine therapy and chemo-endocrine therapy (P = 0.0078). That is, survival rate was superior for the chemoendocrine therapy patients throughout the entire follow-up period. These data suggest that early chemo-endocrine therapy containing Cisplatin, with or without maintenance chemotherapy, is a potentially effective treatment for newly diagnosed metastatic prostate cancer and is worth further investigation via a randomized trial.

Aged↗
PubMed1995

Eicosanoid-mediated Cl- secretion induced by the antitumor drug, irinotecan (CPT-11), in the rat colon.

Irinotecan (CPT-11) is active against a broad range of human cancer. One of the side-effects of irinotecan is a strong diarrhoea. In order to investigate the mechanism underlying this diarrhoea, the effect of irinotecan on anion secretion across the isolated rat distal colon was studied. Irinotecan caused a concentration-dependent increase in short-circuit current (Isc). The increase in Isc was completely dependent on the presence of Cl- ions and was suppressed by furosemide and the Cl- channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), indicating that it is caused by a Cl- secretion. The secretory response was inhibited by indomethacin, 1-benzylimidazole, a thromboxane synthase inhibitor, and SK&F 88046 ((N,N'-bis[7-(3-Chlorobenzeneaminosulfonyl)-1,2,3,4-tetrahydroi soquinolyl) disulfonylimide), a thromboxane A2 receptor blocker. In isolated crypts irinotecan had no effect on the membrane potential. Consequently, the secretion induced by irinotecan is an indirect one, caused by the stimulation of eicosanoid production, e.g. thromboxane A2, in the subepithelial tissue.

Animals↗
PubMed1995

Localization of CD44, the hyaluronate receptor, on the plasma membrane of osteocytes and osteoclasts in rat tibiae.

CD44 is a multifunctional adhesion molecule that binds to hyaluronic acid, type I collagen, and fibronectin. We have studied the immunohistochemical localization of CD44 in bone cells by confocal laser scanning microscopy and transmission electron microscopy in order to clarify its role in the cell-cell and/or cell-matrix interaction of bone cells. In round osteoblasts attached to bone surfaces, immunoreactivity is restricted to their cytoplasmic processes. On the other hand, osteocytes in bone matrices show intense immunoreactivity on their plasma membrane. Intense immunoreactivity for CD44 can be detected on the basolateral plasma membranes of osteoclasts. There is considerably less reactivity observed in the area of the plasma membrane that is in direct contact with bone. The pre-embedding electron-microscopical method has revealed that CD44 is mainly localized on the basolateral plasma membrane of osteoclasts. However, the ruffled border and clear zone show little immunoreactivity. A CD44-positive reaction can be detected on both plasma membranes in the contact region between osteoclasts and osteocytes. These findings suggest that: 1) cells of the osteoblast lineage express CD44 in accordance with their morphological changes from osteoblasts into osteocytes; 2) osteoclasts express CD44 on their basolateral plasma membrane; 3) CD44 in osteoclasts and osteocytes may play an important role in cell-cell and/or cell-matrix attachment via extracellular matrices.

Animals↗
PubMed1995

Functionality of chimeric Rev proteins of HIV/SIV.

Studies on functional compatibility of various Rev proteins derived from all known human and simian immunodeficiency virus subgroups have shown that this essential gene product is not always exchangeable among the viruses. In an attempt to map the region of Rev proteins responsible for the observed nonreciprocal complementation, hybrid genomic Rev expression vectors were constructed by exchanging the first and second exons of rev genes, and were examined for their abilities to activate reporter clones by transfection. With one exception, the second coding exon of rev gene determined the functional specificity of Rev proteins.

Animals↗
PubMed1995

Functional analysis of human spuma retrovirus genome.

Human spuma retrovirus (HSRV) belongs to retroviruses that possess a complex genome organization. HSRV carries at least three extra genes in the region between env and the 3' long terminal repeat, which are not found in simple retroviruses. Via alternative splicing, these HSRV genes can encode several proteins. To genetically study the requirements of these viral proteins for viral replication in tissue cultures, a number of mutant viruses were constructed from an infectious molecular clone of HSRV. All mutants grew normally in the cell lines tested, except for those lacking transcriptional transactivator activity. By reporter-based transient assay systems, no Rev/Rex equivalent was detected in the HSRV proteins.

Alternative Splicing↗
PubMed1995

An immunohistochemical and ultrastructural study of vasomotor nerves in the microvasculature of human dental pulp.

Immunohistochemical methods for S-100 protein and neurone-specific enolase showed two types of nerve endings around the pulp microvasculature: the free endings, which comprise the major neural component and are distributed in all types of microvessel such as arterioles, venules and capillaries; and the varicose endings. The varicose ending was a less frequent, minor component observed only in the arterioles. Both immunohistochemical and ultrastructural observations confirmed that the varicose endings were the terminal axons of efferent vasomotor nerves. Further extensive ultrastructural examinations on the vasomotor nerves added the following new findings to our previous reports. Vasomotor nerves sometimes ramified into more than two terminal axons around arterioles, and most of these ramified axons ended in the adventitia-media junction of the arteriolar wall; however, nerve endings occasionally penetrated into the media. These findings suggest an intimate structural association between vasomotor nerves and arterioles in regulating the arteriolar microcirculation in human dental pulp by stimulating smooth-muscle cells not only of the outermost but also of the inner layers. Furthermore, the deep penetration of terminal axons into the arteriolar wall seems to provide effective regulation of pulpal blood flow under physiological and pathological conditions.

Adult↗
PubMed1995

Immunohistochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth.

The condition of the pulp tissue was classified into seven groups according to the depth of carious lesions from stage (S) 0 (non-carious teeth) to S6 (exposed pulp). A substantial change in the infiltration of immunocompetent cells occurred between S3 and S4; all types were markedly increased in S4 as compared to S3, with a remarkable increase in the number of helper T lymphocytes, B-lineage cells, neutrophils and macrophages. Therefore, the pulpal immune reaction to carious stimuli could be classified into early (S1-S3) and advanced phases (S4-S6). In the early phase a cellular immunoresponse would be induced by T-lineage cells, and in the advanced phase the humoral immunoresponse is furthered by B-lineage cells concomitant with the destruction of pulp tissue by proteolytic enzymes released from infiltrating neutrophils and macrophages. Human dental pulp is thus equipped with a functional immune response that is sufficient as a biodefensive mechanism. Dental caries should be treated before S4.

Adult↗
PubMed1995

The proliferative activity in oral epithelial dysplasia analyzed by proliferating cell nuclear antigen immunostaining and argyrophilic nucleolar organizer region staining.

The proliferative activity of leukoplakia without dysplastic change (LP), epithelial dysplasia (ED), and squamous cell carcinoma (SCC) in the oral mucosa was examined by means of proliferating cell nuclear antigen (PCNA) immunostaining, silver-binding argyrophilic nucleolar organizer region (AgNOR) staining, and the frequency of mitotic figures. Significant differences in the labeling index of PCNA immunostaining (PI) and mitotic index (MI) were noted between LP and ED and between ED and SCC. The mean numbers of AgNORs (AI) significantly differed between ED and SCC. There was a significant positive correlation between PI and MI in samples of ED. However, there was no significant correlation between AI and other indexes. The number and the distribution of PCNA-positive cells in ED varied among samples. Five samples with higher PI and MI indexes than the mean values were selected from those of dysplasia based on the correlation between PI and MI. Their histological features symptomatic of oral ED as defined by the World Health Organization (WHO) Collaborating Centre in 1978, were investigated and compared with 10 samples with lower indexes. Histological findings, such as "loss of polarity of the basal cells," "an increased nuclear-cytoplasmic ratio," "cellular pleomorphism," and "enlarged nucleoli," were significant histological features of these five samples. This study showed that the four histological components described previously and the increased number of mitotic figures used as the index of proliferating activity were the main histological components related to severe ED of oral mucosa. They will provide a useful means of deciding the histopathological grade of oral ED.

Adult↗
PubMed1995

Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces.

Poly(N-isopropylacrylamide) (PIPAAm), exhibiting a lower critical solution temperature (LCST) at 25 degrees C in physiological phosphate buffered saline solution (pH 7.4) and at 32 degrees C in pure water, was grafted onto the surfaces of commercial polystyrene cell culture dishes. This PIPAAm-grafted surface exhibited hydrophobic surface properties at temperatures over the LCST and hydrophilic surface properties below the LCST. Endothelial cells and hepatocytes attached and proliferated on PIPAAm-grafted surfaces at 37 degrees C, above the LCST. The cultured cells were readily detached from these surfaces by lowering the incubation temperature without the usual damage associated with trypsinization. In this case, the optimum temperature for cell detachment was 10 degrees C for hepatocytes and 20 degrees C for endothelial cells. Cell detachment was partially inhibited by sodium azide treatment, suggesting that cell metabolism directly affects cell detachment. Morphological changes of the adherent cells during cell detachment experiments indicated further involvement of active cellular metabolic processes. Cells detached from hydrophobic-hydrophilic PIPAAm surfaces not only via reduced cell-surface interactions caused by the spontaneous hydration of grafted PIPAAm chains, but also by active cell morphological changes which were a function of cell metabolism.

Acrylic Resins↗