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Biomedical subjects

Y Sakai

Publications and source records attributed to Y Sakai.

At least 271 records · Page 15Linked to original sources
PubMed1999

Production of secretory component and pathogenesis of gastric cancer in Helicobacter pylori-infected stomach.

The purpose of this study was to examine the production of secretory component (SC) and immunoglobulin A (IgA) in the gastric mucosa with Helicobacter pylori infection and to investigate the influence of immunological reactions on various phases of infection (gastritis, intestinal metaplasia, gastric cancer). Production of SC and IgA was assessed by immunohistochemical staining in (1) endoscopic biopsy samples of H. pylori-eradicated cases (n = 25), and (2) surgically resected stomach tissues of H. pylori-positive gastric cancer cases, intestinal type (IGC, n = 25) and diffuse type (DGC, n = 25). Before eradication therapy, all samples showed positive staining of SC and IgA in epithelial cells, and IgA was also positive in plasma cells in the mucosal layer. H. pylori bacteria were positively stained for SC and IgA. After treatment, the degree of SC and IgA staining in epithelial cells was reduced with successful eradication; but with intestinal metaplasia, SC staining was positive regardless of the results of treatment. In nonmetaplastic mucosa, SC-positive cells were increased in the glandular neck zone to the surface mucosal layer; and the intensity of SC staining was increased in proportion to the degree of mucosal inflammation and IgA-positive cell aggregation. In intestinal metaplasia, SC was positive irrespective of the degree of inflammation. Most cancer foci also showed positive staining of SC, irrespective of histological type. Production of SC and IgA was thought to be a specific reaction against H. pylori infection, occurring from the early to the late stages and not limited to intestinal metaplasia. It was suggested that immunological reactions against H. pylori infection might generally be involved with the pathogenesis of intestinal metaplasia and both histological types of gastric cancer (IGC and DGC).

Gastric Mucosa↗
PubMed1999

Extended thymectomy for myasthenia gravis in an octogenarian. A case report.

An 86-year-old woman with myasthenia gravis successfully underwent an extended thymectomy. The patient had a 2-year history of generalized myasthenia and had limited response to anticholinesterases and steroids. An extended thymectomy was successfully performed in the usual fashion. The postoperative course was uneventful except for an episode of psychosis probably due to postoperative steroid therapy. The steroid dosage was gradually reduced to 5 mg/day over seven months after the operation, during which she became fully asymptomatic. No symptoms due to myasthenia have surfaced for over 2 years after surgery. Although elderly patients are usually considered to be less responsive to an operation, thymectomy may sometimes be the treatment of choice for myasthenia gravis even in octogenarians.

Aged↗
PubMed1998

Regulation of peroxisomal proteins and organelle proliferation by multiple carbon sources in the methylotrophic yeast, Candida boidinii.

A methylotrophic yeast, Candida boidinii, was grown on various combinations of peroxisome-inducing carbon source(s) (PIC(s)), i.e. methanol, oleate and D-alanine, and the regulation of peroxisomal proteins (both matrix and membrane ones) and organelle proliferation were studied. This regulation was followed (1) at the protein or enzyme level by means of the peroxisomal enzyme activity and Western analysis; (2) at the mRNA level by Northern analysis; and (3) at the organelle level by direct observation of peroxisomes under a fluorescent microscope. Peroxisomal proliferation was followed in vivo by using a C. boidinii strain producing a green fluorescent protein having peroxisomal targeting signal 1. When multiple PICs were used for cell growth, C. boidinii induced specific peroxisomal proteins characteristic of all PIC(s) present in the medium, responding to all PIC(s) simultaneously. Thus, these PICs were considered to induce peroxisomal proliferation independently and not to repress peroxisomes induced by other PICs. Next, the sensitivity of the peroxisomal induction to glucose repression was studied. While the peroxisomal induction by methanol or oleate was completely repressed by glucose, the D-alanine-induced activities of D-amino acid oxidase and catalase, Pmp47, and the organelle proliferation were not. These results indicate that peroxisomal proliferation in yeasts is not necessarily sensitive to glucose repression. Lastly, this regulation was shown to occur at the mRNA level.

Acyl-CoA Oxidase↗
PubMed1998

Selection of DNA binding sites for human transcriptional regulator GATA-6.

The DNA-binding sites for transcriptional regulator GATA-6 were selected and amplified by means of a PCR-mediated random-site selection method involving filter binding and gel-mobility shift analysis using the zinc finger region of human GATA-6 fused with GST. Sequencing and comparison of the selected clones suggested that (A/T/C)GAT(A/T)(A) is the consensus binding sequence, which is similar to the GATA motif (A/T)GATA(A/G) and the gastric motif (G/C)PuPu(G/C)NGAT(A/T)PuPy. GATA-6 also binds to a similar sequence containing the GATC but not the GATG sequence. These results indicated that GATA-6 shows broader sequence specificity as to DNA binding. However, adenine is favored on both sides of the core for strong binding [AGAT(A/T)A], and the order of binding is GATA > GATT > GATC. Full-length human GATA-6 expressed in COS-1 cells showed essentially the same binding specificity, suggesting that the zinc finger region of GATA-6 mainly contributes to the selection of the binding sequence. Furthermore, the non-RI method presented in this paper is convenient for determination of the binding sites of other DNA-binding proteins.

Binding Sites↗
PubMed1998

The mouse Sox5 gene encodes a protein containing the leucine zipper and the Q box.

The nucleotide sequence of mouse Sox5 (mSox5) cDNA derived from the testis has been reported by Denny et al. (EMBO J. 11 (1992) 3705-3712). We newly isolated an mSox5 cDNA derived from 8.5-day mouse embryo. Our cDNA encodes a protein of 763 amino acids, which is considerably larger in size than the previous one (392 amino acids) derived from the adult mouse testis. The most significant difference between the embryo-derived and testis-derived mSox5 cDNAs is that the embryonic one encodes a leucine zipper motif and a neighboring glutamine-rich sequence stretch (named Q box), but the testis-derived one does not. The leucine zipper and the Q box are highly conserved among type-D SOX proteins including mSox5. mSox5 was suggested to be a single-copy gene by Southern analysis. With reverse transcription/polymerase chain reaction, we found that not only mouse embryo, but also the adult mouse testis, express mSox5 mRNA species encoding the leucine zipper and the Q box.

Amino Acid Sequence↗
PubMed1998

Lower expression of neutrophil adhesion molecule indicates less vessel wall injury and might explain lower restenosis rate after cutting balloon angioplasty.

BACKGROUND: The Cutting Balloon is a novel dilatation catheter for coronary angioplasty (InterVentional Technologies Inc). It produces longitudinal, microsurgical incisions in the vessel wall before the actual dilatation. It is assumed that these controlled surgical incisions relieve hoop stress and reduce vessel wall injury and eventually restenosis. However, no clinical indicator to support the theory of reduced injury has been described. Certain clusters of differentiation (eg, CD11, CD18 on the leukocytes) are implicated in leukocyte adhesion, increased permeability, and opsonization. Therefore, they might serve as clinical indicators of the injury level of the vessels after angioplasty. METHODS AND RESULTS: We randomly selected 64 patients with isolated left anterior descending coronary artery disease for either Cutting Balloon angioplasty or conventional balloon angioplasty. The expression of CD18 and CD11b on the surface of neutrophils was determined by flow cytometric analysis. Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) were also measured. The expression of both the CD18 and CD11b in the coronary sinus blood gradually increased and reached its maximum at 48 hours after angioplasty. The sICAM-1 levels in the coronary sinus serum also increased after angioplasty. Percentage increases of CD18 and CD11b expression and the increase of the sICAM-1 levels at 48 hours after angioplasty (as ratios to baseline values before angioplasty) were less in the Cutting Balloon angioplasty group than in the conventional balloon angioplasty group (CD18, 1.10+/-0.05 versus 1.31+/-0.05, P<0.05; CD11b, 1.23+/-0.06 versus 1.72+/-0.10, P<0.001; sICAM-1, 1.12+/-0.05 versus 1.25+/-0.02, P<0.05). In all patients, the late lumen loss at follow-up angiogram positively correlated with the increased levels of CD11b (R=0.59, P<0.001) and sICAM-1 (R=0.38, P<0.05) at 48 hours after angioplasty. CONCLUSIONS: Balloon angioplasty upregulated Mac-1 (CD11b/CD18) on the surface of the neutrophils and increased sICAM-1 levels in association with late loss increase. These changes were significantly smaller in the Cutting Balloon angioplasty group than in the conventional balloon angioplasty group. This suggests that Cutting Balloon angioplasty may produce less vessel wall injury and, consequently, less neutrophil activation, which may account for the lower rate of restenosis.

CD18 Antigens↗
PubMed1998

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1alpha (SDF-1alpha) and SDF-1beta are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage.

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1alpha (SDF-1alpha) and 1beta (SDF-1beta), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34(+) progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1alpha confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1alpha and SDF-1beta likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4(+) cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1alpha and SDF-1beta are involved.

Animals↗
PubMed1998

Peroxisome degradation by microautophagy in Pichia pastoris: identification of specific steps and morphological intermediates.

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl ) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor-product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.

Adaptation, Physiological↗
PubMed1998

Molecular cloning and characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein).

We have isolated a cDNA clone encoding a germ cell specific protein from an expression cDNA library prepared from the mouse testis, using testis-specific polyclonal antibodies. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands for male meiotic metaphase chromosome-associated acidic protein is proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.

Amino Acid Sequence↗
PubMed1998

Two novel testicular serine proteases, TESP1 and TESP2, are present in the mouse sperm acrosome.

To identify a novel candidate(s) for acrosomal proteins that act on the sperm/egg interaction, a DNA fragment was PCR-amplified from a cDNA library of acrosin-deficient mouse testis and then used as a probe to screen a mouse testis cDNA library. Complementary DNA clones encoding each of two similar but different serine proteases, TESP1 and TESP2, have been identified. The nucleotide sequences of these clones indicate that mouse TESP1 and TESP2 are initially synthesized as preproproteins of 367 and 366 amino acids, respectively. Comparison of the two TESP sequences with those of typical serine proteases suggests that each TESP zymogen is probably converted into a two-chain mature enzyme consisting of light and heavy chains covalently linked by a single pre-existing disulfide bond. The conversion may be accomplished by another protease(s) with a trypsin-like cleavage specificity, since it is unlikely that the mature TESP1 and TESP2 are capable of splitting the Lys-Ile bond between the light and heavy chains. Northern blot analysis of total cellular RNA demonstrates that the TESP1 and TESP2 genes are expressed only in the testis, and the transcripts are abundantly present in the haploid round spermatids. Moreover, immunocytochemical analysis of mouse cauda epididymal sperm using affinity-purified antibodies reveals that these two TESPs are both localized in the sperm acrosome and are released during the acrosome reaction induced by calcium ionophore A23187. These findings provide additional clues for elucidating the mechanisms involved in the sperm/egg interactions, including penetration of the zona pellucida by sperm.

Acrosome↗
PubMed1998

Importance of the N-glycan in the V3 loop of HIV-1 envelope protein for CXCR-4- but not CCR-5-dependent fusion.

The V3 region of HIV-1 envelope protein possesses a single N-linked sugar chain, which is conserved in most HIV-1 strains. We studied its role in the life cycle of HIV-1 strains with different co-receptor usage. Removal of the glycan appeared to cause a marked reduction of CXCR-4- but not CCR-5-dependent virus entry. A basic amino acid substitution at the 11th position of V3 markedly compensated for the removal of the N-glycan. These results indicate that the N-glycan plays an important role for CXCR-4-dependent virus entry and that this role is exerted in a particular context of the peptide backbone.

Amino Acid Sequence↗
PubMed1998

Large quantity production with extreme convenience of human SDF-1alpha and SDF-1beta by a Sendai virus vector.

We describe a robust expression of human stromal cell-derived factor-1alpha (SDF-1alpha) and SDF-1beta, the members of CXC-chemokine family, with a novel vector system based upon Sendai virus, a non-segmented negative strand RNA virus. Recombinant SDF-1alpha and SDF-1beta were detected as a major protein species in culture supernatants, reached as high as 10 microg/ ml. This remarkable enrichment of the products allowed us to use even the crude supernatants as the source for biological and antiviral assays without further concentration nor purification and will thus greatly facilitate to screen their genetically engineered derivatives.

Anti-HIV Agents↗
PubMed1998

Enhanced and specific gene expression via tissue-specific production of Cre recombinase using adenovirus vector.

A tissue-specific promoter is potentially valuable for the study of specific gene function and for gene therapy, as it permits a linked cytotoxic or any other gene to be expressed specifically in target cells. The expression levels of such promoters are generally low, and we have therefore developed a novel and general method to enhance the expression level of a tissue-specific promoter while maintaining specificity. We constructed a "regulator" recombinant adenovirus (rAd) producing the site-specific recombinase Cre under the control of the hepatocarcinoma-specific alpha-fetoprotein (AFP) promoter. The rAd was infected to AFP-producing cells together with a "target" rAd containing a Cre-activating potent expression unit. In in vitro experiments, the double infection method gave about 50-fold higher expression than the single rAd infection directly driven by the AFP promoter, while maintaining strict specificity to AFP-producing cells. The enhanced and specific expression was also observed in in vivo tumor models. This method may contribute not only to the establishment of specific gene therapies but also to basic study for elucidating cell-type specific gene functions.

Adenoviridae↗
PubMed1998

Isolation and expression of a human SRY-related cDNA hSOX20.

SOX is a family of genes related to the testis-determining gene, SRY. We have isolated and sequenced an hSOX20 cDNA from a cell line of human embryonic carcinoma. This cDNA contains an open reading frame (ORF) encoding 233 amino acids. The protein encompasses an SRY-type HMG box exhibiting strong homologies to those of mouse Sox15 and Sox16. Various adult and fetal tissues were tested for hSOX20 mRNA by Northern analysis. Its expression is restricted to the fetal testis and the size of the transcript is 1.5 knt. Electrophoretic mobility shift assay indicated that recombinant hSOX20 polypeptide is capable of binding to AACAAT sequence.

Adult↗
PubMed1998

Circadian variation of myocardial ischemia in patients with unstable angina pectoris secondary to fixed and/or spastic coronary narrowing.

We investigated the circadian variation of myocardial ischemia in patients with unstable angina using a 24-hour 12-lead electrocardiographic monitoring system with computer analysis. The circadian variation of ischemic attacks in patients who had ST elevation showed a similarity to that of the onset of acute myocardial infarction and most of these patients had vasospasm without organic stenosis; no similarity was seen in patients with organic stenosis.

Aged↗
PubMed1998

Cloning and characterization of mouse mSox13 cDNA.

A novel SRY-related cDNA, mSox13, was isolated from a lambda phage library derived from mouse embryo. The cDNA encodes a protein of 595 amino acids containing the SRY-type high mobility group (HMG) box and a putative leucine zipper motif. A sequence comparison of mSox13 and other type-D SOX proteins shows that the leucine zipper and a neighboring glutamine-rich sequence stretch, which was named Q box, are well conserved among known type-D SOX proteins. The expression of mSox13 is restricted to the kidney and ovary. The electrophoretic mobility shift assay indicates that the recombinant mSox13 protein is capable of binding to the AACAAT sequence.

Amino Acid Sequence↗
PubMed1998

Regulation of expression of human intestinal bile acid-binding protein in Caco-2 cells.

Molecular mechanisms of the bile acid active transport system in the ileal enterocytes remain unknown. We examined whether bile acids affect human enterocyte gene expression of intestinal bile acid-binding protein (I-BABP), a component of this transport system. Differentiated Caco-2 cells were incubated in the presence of human bile, bile acids or other lipids. The level of I-BABP expression was evaluated by Northern and Western blot analyses. A 24 h incubation of Caco-2 cells in a medium containing either bile or bile acids resulted in a remarkable 7.5-fold increase in the I-BABP mRNA level over the control level. Neither cholesterol, palmitic acid, phosphatidylcholine nor cholestyramine treated bile showed any difference in I-BABP mRNA expression from the control. Bile acid treatment increased the level of I-BABP mRNA in Caco-2 cells in a time- and dose-dependent manner. Western blot analysis showed that this induction led to increase in cytosolic I-BABP. Chenodeoxycholic acid and deoxycholic acid showed greater induction effects than other hydrophilic bile acids, including their own glycine conjugates. Pretreatment by actinomycin D or cycloheximide completely inhibited the up-regulation of I-BABP expression by bile acid. Bile acids, especially lipophilic bile acids, increase the I-BABP expression in Caco-2-cells, suggesting that luminal bile acids play an important role in regulating the I-BABP gene expression.

Bile Acids and Salts↗