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Biomedical subjects

Y Tamai

Publications and source records attributed to Y Tamai.

At least 55 records · Page 3Linked to original sources
PubMed1999

A monoclonal antibody against a glycolipid SEGLx from Spirometra erinaceieuropaei plerocercoid.

A mouse monoclonal antibody AK97 (IgM) was established against a new type of glycosphingolipid, SEGLx, isolated from plerocercoids of tapeworm, Spirometra erinaceieuropaei. The chemical structure of SEGLx (Gal beta1-4(Fuc alpha1-3)(Glc beta1-3Gal beta1-ceramide) had been previously characterized. The specificity of AK97 was determined by thin-layer chromatography-immunostaining and enzyme-linked immunosorbent assay. AK97 was found to be directed to SEGLx and GalSEGLx (Gal beta1-4(Fuc alpha1-3)Glc beta1-3(Gal beta1-6)Gal beta1-ceramide) and also showed cross-reactivity with the stage specific embryonic antigen-1 (SSEA-1), the epitope being defined to be the non-reducing terminal trisaccharide sequence. On immunohistochemical examination, AK97 predominantly stained the tegument, the external surfaces of worms which have a brush border-like organization. Based on the immunohistochemical findings for the staining liability as to organic solvents and the results of Western blot analysis of the plerocercoid glycoproteins, it was proved that the antigens in the tapeworm were glycolipids. Considering that the tapeworm is in direct contact with its host's tissue through the tegument, the membrane surface of which is exposed to the external environment, it is suspected that SEGLx and GalSEGLx on the tegument play functionally important roles in the host parasite interaction.

Animals↗
PubMed1999

Colonic hamartoma development by anomalous duplication in Cdx2 knockout mice.

To determine the biological role of caudal-like homeobox gene CDX2, we constructed knockout mice in which its mouse homologue Cdx2 was inactivated by homologous recombination, placing a bacterial lacZ gene under the control of the Cdx2 promoter. Although the homozygous mutants died in utero around implantation, the heterozygotes were viable and fertile and expressed lacZ in the caudal region in early embryos and in the gut tissues in adults. The heterozygotes developed cecal and colonic villi by anteriorization and formed hamartomatous polyps in the proximal colon. The hamartoma started to develop at 11.5 days of gestation as an outpocket of the gut epithelium, which ceased to express the remaining Cdx2 allele. The outpocket then expanded as a partially duplicated gut but was contained as a hamartoma after birth. In adult mice, these hamartomas grew very slowly and took a benign course. None of them progressed into invasive adenocarcinomas, even at 1.5 years of age. Whereas the cecal and colonic villi expressed lacZ, the hamartoma epithelium did not, nor did it express Cdx2 mRNA from the wild-type allele. However, genomic DNA analysis of the polyp epithelium did not show a loss of heterozygosity of the Cdx2 gene, suggesting a mechanism of biallelic Cdx2 inactivation other than loss of heterozygosity. These results indicate that the Cdx2 haploin-sufficiency caused cecal and colonic villi, whereas the biallelic inactivation of Cdx2 triggered anomalous duplications of the embryonic gut epithelium, which were contained as hamartomas after birth.

Animals↗
PubMed1999

Isolation and characterization of the ATF2 gene encoding alcohol acetyltransferase II in the bottom fermenting yeast Saccharomyces pastorianus.

The ATF2 gene encodes alcohol acetyltransferase II, which catalyses the synthesis of isoamyl acetate from acetyl coenzyme A and isoamyl alcohol. To characterize the ATF2 gene from the bottom fermenting yeast Saccharomyces pastorianus, the S. pastorianus ATF2 gene was cloned by colony hybridization using the S. cerevisiae ATF2 gene as a probe. When an atf1 null mutant strain was transformed with a multi-copy plasmid carrying the S. pastorianus ATF2 gene, the AATase activity of this strain was increased by 2.5-fold compared to the control. The S. pastorianus ATF2 gene has 99% nucleic acid homology in the coding region and 100% amino acid homology with the S. cerevisiae ATF2 gene. Southern blot analysis of chromosomes separated by pulse-field gel electrophoresis indicated that the ATF2 gene probe hybridized to chromosome VII in S. cerevisiae and to the 1100 kb chromosome in S. pastorianus. As S. pastorianus is thought to be a hybrid of S. cerevisiae and S. bayanus, the S. bayanus-type gene, which has a relatively low level of homology with the S. cerevisiae-type gene, is also usually detected. Interestingly, an S. bayanus-type ATF2 gene could not be detected. These results suggested that the cloned ATF2 gene was derived from S. cerevisiae. Analysis using an ATF2-lacZ fusion gene in S. pastorianus showed that expression of the ATF2 gene was relatively lower than that of the ATF1 gene and that it is repressed by aeration but activated by the addition of unsaturated fatty acids. The S. pastorianus ATF1, Lg-ATF1 and ATF2 Accession Numbers in the DDBJ Nucleotide Sequence Database are D63449, D63450 and D86480, respectively.

Acetyltransferases↗
PubMed1999

A novel hemiacetal dehydrogenase activity involved in ethyl acetate synthesis in Candida utilis.

Acetate ester synthesis was studied in vitro with the ethyl acetate-producing yeast Candida utilis. The level of enzyme activity observed for the NAD+-dependent hemiacetal dehydrogenase acting on hemiacetal, which was produced non-enzymatically from an alcohol and an aldehyde, was much greater than that for the other enzyme involved in ester synthesis, alcohol acetyltransferase. The level of ethyl acetate synthesis in vivo approximately paralleled the hemiacetal dehydrogenase (HADH) activity. The results suggest that the main pathway for ethyl acetate synthesis in C. utilis involves a novel hemiacetal dehydrogenase activity.

Journal Article↗
PubMed1999

Characterization of the Na+-ATPase gene (ZENA1) from the salt-tolerant yeast Zygosaccharomyces rouxii.

In order to clarify the relationship between the salt tolerance of Zygosaccharomyces rouxii and the function of Na+-ATPase, a gene which exhibited homology to the Na+-ATPase gene (ZENA1) of Saccharomyces cerevisiae was isolated from Z. rouxii. This newly isolated gene (ZENA1) encoded a product of 1048 amino acids. The predicted amino-acid sequence of Zena1p was highly homologous to that of S. cerevisiae Ena1p and Ena2p, and Schwanniomyces occidentalis Ena1p and Ena2p, but showed low homology to that of Zpma1p, which is the product of the Z. rouxii plasma membrane H+.ATPase gene (ZENA1). Zena1p shares the peptide motifs which have been suggested to participate in the function of ATPase. Expression of ZENA1 was observed, but was independent of NaCl shock. When ZENA1 was expressed in salt-sensitive S. cerevisiae under the regulation of a GAL1 promoter by using the expression vector pYES2, salt tolerance of the transformants was observed. The growth characteristics of Zena1Delta-disruptants of Z. rouxii and the pH profiles of their plasma membrane ATPase activity were almost the same as those of the wild-type strain, indicating that the function of Zena1p is of little relevance to the salt tolerance property of Z. rouxii. By considering the close relationship between the salt tolerance of Z. rouxii and the function of its Na+/H+-antiporter, we concluded that the extrusion of Na+ across the plasma membrane in Z. rouxii cells might be carried out mainly via the function of the Na+/H+-antiporter in a high salinity environment.

Journal Article↗
PubMed1999

Induction of apoptosis in HL-60 cells by skimmed milk digested with a proteolytic enzyme from the yeast Saccharomyces cerevisiae.

Bovine skimmed milk digested with cell-free extract of the yeast Saccharomyces cerevisiae was found to exhibit proliferation inhibition activity towards human leukemia (HL-60) cells. The optimum pH for digestion of skimmed milk and production of the proliferation inhibition factor was pH 4.8. Nondigested skimmed milk exhibited little suppressive effect on the proliferation of HL-60 cells. An active enzyme involved in the production of cell proliferation inhibitory materials from skimmed milk was purified from the cell-free extract of S. cerevisiae by a series of column chromatographies: DEAE-Sephacel, D-tryptophan methyl ester-Sepharose 4B, Hiload Superdex G-200 and HPLC Mono Q. The homogeneous purified enzyme and exhibited a molecular mass of 33 kDa in sodium dodeceyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was identified as protease B by N-terminal amino acid sequence analysis. Bovine skimmed milk digested with purified protease B was found to inhibit proliferation activity of HL-60 cells most strongly when digestion was conducted at pH 4.8. The cell proliferation inhibition activity induced by digested skimmed milk was shown to be due to the induction of apoptosis, demonstrated by the formation of apoptotic bodies and fragmentation of DNA in treated cells. The proliferation inhibition factors produced were recovered in the soluble fraction of 92% ethanol, suggesting that the factors were hydrophilic low molecular mass substances derived from skimmed milk.

Journal Article↗
PubMed1999

Gene structure and amino acid sequence of Latimeria chalumnae (coelacanth) myelin DM20: phylogenetic relation of the fish.

The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2-7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941-1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.

Amino Acid Sequence↗
PubMed1999

Retrovirus integration site Mintb encoding the mouse homolog of hnRNP U.

Retroviral genes are not usually expressed in mouse embryonal carcinoma (EC) cells, but they are readily expressed upon differentiation of these cells. We previously reported the isolation of EC cell lines that express a neomycin resistance (neo) gene introduced by a recombinant transducing Moloney murine leukemia virus from specific integration sites, Minta, Mintb, Mintc, or Mintd. In some of these clones, the entire 5' long terminal repeat (LTR) was deleted, and the neo gene was expressed by read-through transcription from upstream cellular promoters in a "promoter-trap" fashion. One such promoter ("promoter B" at the Mintb locus) was found in a CpG island, associated with an upstream enhancer ("enhancer B"). Although enhancer B caused expression of the neo gene in the transductant EC cell line, no endogenous transcription from promoter B was detected in the parental EC or NIH3T3 cells. In contrast, we found a strong counter-flow endogenous transcription unit ("R" for reverse), which apparently interfered with transcription from promoter B. Promoter R turned out to have a bidirectional activity in transfection assays. In normal tissues, promoter R activates gene R, which encodes an 800-residue protein that is highly homologous to the rat and human heterogeneous nuclear ribonucleoprotein U (hnRNP U). Northern and in situ hybridization analyses revealed that gene R was abundantly expressed in the testis, especially in the pachytene spermatocytes and round spermatids.

3T3 Cells↗
PubMed1999

Effects of polymer chains on structure and dynamics of supercooled water in poly(vinyl alcohol).

Molecular-dynamics simulations for poly(vinyl alcohol) hydrogels have been carried out in a wide temperature range 150-400 K to examine effects of polymer chains on structure and dynamics of supercooled water in hydrogels. A fragile-strong character of water under perturbations of polymer chains is examined. Polymer chains affect the fragile-strong character of water, which is manifested in temperature dependences of potential energy, translational diffusion coefficient, and orientational relaxation time. Water coexisting with polymer has a rather stronger character in the strong-fragile classification than that in pure water. Structure and dynamics of water in hydrogels are significantly altered by the slow dynamics of polymer chains, as well as the structure and dynamics of hydrogen-bond networks.

Journal Article↗
PubMed1999

Comparison of the effects of acetylsalicylic acid, ticlopidine and cilostazol on primary hemostasis using a quantitative bleeding time test apparatus.

We examined and compared the effects of aspirin (ASA), ticlopidine (TP) and cilostazol (CS) on bleeding time (BT) in 10 healthy adult male subjects using a newly developed quantitative bleeding time (QBT) test apparatus capable of simultaneously measuring total blood loss (Tv), maximum bleeding rate (Rmax), and bleeding pattern in addition to BT. All 3 drugs inhibited platelet aggregation response to ADP, collagen, epinephrine and arachidonic acid (p < 0.05), but not to ristocetin. Following oral administration of ASA (330 mg/day) or TP (300 mg/day) for 3 days, BT was significantly prolonged (mean BT increased from 359.3 to 646.0 s, p < 0.001, and from 323.3 to 528. 7 s, p < 0.01, respectively) and Tv was significantly increased (from 14.5 to 30.2 microl, p < 0.05, and from 12.5 to 19.2 microl, p < 0.01, respectively). Aspirin also increased Rmax (from 0.118 to 0. 159 microl/s, p < 0.05). The prolonged bleeding patterns after administration of ASA and TP were both type III, which has been reported to be less likely to lead to bleeding accidents. In contrast, none of these QBT parameters were altered by CS administration.

Adult↗
PubMed1999

Purification and characterization of phospholipase B from Kluyveromyces lactis, and cloning of phospholipase B gene.

Phospholipase B (PLB) from the yeast Kluyveromyces lactis was purified to homogeneity from culture medium. The enzyme was highly glycosylated with apparent molecular mass of 160-250 kDa, and had two pH optima, at pH 2.0 and pH 7.5. At acidic pH the enzyme hydrolyzed all phospholipid substrates tested here without metal ion. On the other hand, at alkaline pH the enzyme showed substrate specificity for phosphatidylcholine and lysophosphatidylcholine and required Ca2+, Fe3+, or Al3+ for the activity. The alkaline activity was increased more than 20-fold in the presence of Al3+ compared to that in the presence of Ca2+. cDNA sequence of PLB (KlPLB) was analyzed by a combination of several PCR procedures. KlPLB encoded a protein consist of 640 amino acids and the deduced amino acid sequence showed 66.7% similarity with the T. delbrueckii PLB. The amino acid sequence contained the lipase consensus sequence (G-X-S-X-G) and the catalytic aspartic acid motif. Replacement of Arg-112 or Asp-406 with alanine caused loss of the enzymatic activity at both pH. These results suggested that PLB activity are dependent on a catalytic mechanism similar to that of cytosolic phospholipase A2.

Acyltransferases↗
PubMed1999

[Rapidly progressive irradiated cervical cancer that metastasized to the liver during therapy for idiopathic thrombocytopenic purpura].

A 37-year-old woman was given a diagnosis of cervical cancer in August 1994. Because of severe thrombocytopenia, she was given radiation therapy at 50 Gy with great effectiveness. The thrombocytopenia was diagnosed as idiopathic thrombocytopenic purpura. Because the patient refused to undergo a splenectomy operation, she was treated with prednisolone, gamma-globulin, and danazol with no effect. In January 1995 she began receiving azathioprine and her platelet count gradually increased. In March, she complained of severe left abdominal pain but abdominal computed tomography (CT) scans showed no abnormal findings. Nonetheless, the patient's lumbago persisted and her liver dysfunction was progressive. Abdominal CT scans performed on April 18 disclosed multiple liver tumors. The patient died on April 28. Autopsy revealed that the cervical cancer was the primary origin of the liver tumors. We concluded that extra precautions should be taken when administering immunosuppressive therapy to patients with a history of malignant diseases.

Adult↗
PubMed1999

Vaginal applicators (ovoids) for local control and alleviation of rectal complications of cervical cancers treated by brachytherapy.

PURPOSE: To assess the effects of vaginal applicators (ovoids) on dose distribution, local control and rectal complications in cervical cancers treated by brachytherapy. METHODS: From 1984 to 1992, 41 patients (15 stages I+II; 26 stages III+IV) were treated by high-dose-rate brachytherapy (20 Gy in 4 fractions) after external beam irradiation (36-56 Gy). Twenty-three patients were treated by standard application of both intrauterine tandem and ovoids, and 18 patients were treated by non-standard application. RESULTS: The five-year, local-relapse-free rates by standard application and tandem alone were 69 and 65% overall; 83 and 100% for stages I+II; and 62 and 49% for stages III+IV. Local control was related to tumor response following the external beam irradiation and initial tumor size by multivariate analysis. Rectal complications seen in patients followed for more than 1 year were 33% after standard application and 22% for tandem use alone. The rectal doses given for standard application (24 Gy) and tandem use (16 Gy) were significantly different. CONCLUSIONS: The ovoid sources did not always contribute to local control, and occasionally led to rectal complications. The optimization of brachytherapy was dependent on patients' anatomy, tumor size, and tumor response.

Adult↗
PubMed1999

Nurses' perspectives concerning do-not-resuscitate (DNR) orders.

The purpose of this study was to investigate the views of the nursing staff concerning do-not-resuscitate (DNR) orders at the Tokai University Hospital where a controversial incident occurred several years ago. A 'Questionnaire on DNR Orders' was circulated and the anonymous answers were collected two weeks later. The questionnaire was returned by 706 of 780 (90.5%) nurses from every ward/specialty, which revealed that 87% of the nurses felt that DNRs were occasionally necessary, with more than 40% of the nurses answering that they took part in DNR. Further, 36% of the nurses stated that patient consent was indispensable, and 64% thought that the patient's family and physician could decide DNR in the event the patient was physically unable to give consent. Moreover, 66% of the nurses expected the establishment of a DNR order sheet to be formulated as a matter of hospital policy; only 5% of the nurses thought that such an order sheet would not be necessary. Comparing these results with a previous study polling physicians at the Tokai University Hospital, nurses are more likely than physicians to think that patient consent is indispensable, and want the establishment of a standardized DNR order sheet as hospital policy. There is, in fact, a "tacit understanding" between physicians and patients' families in medical practice in Japan. However, DNR is definitely a medical decision. Therefore it should be clearly stated in a standardized format, although such a procedure presently seems unlikely, in view of the Japanese traditional value system.

Adult↗
PubMed1998

A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

ADAM Proteins↗
PubMed1998

An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3.

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCzeta and PKClambda have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype-specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKClambda to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.

3T3 Cells↗
PubMed1998

Characterization of a second gene (ZSOD22) of Na+/H+ antiporter from salt-tolerant yeast Zygosaccharomyces rouxii and functional expression of ZSOD2 and ZSOD22 in Saccharomyces cerevisiae.

We reported in our previous paper on the characterization of the Na+/H(+)-antiporter gene (ZSOD2) closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii. In the present paper, we have cloned a second gene (ZSOD22) of Na+/H+ antiporter from Z. rouxii. The deduced amino acid sequence of Zsod22p was highly homologous to that of Zsod2p, Sod2p from Schizosaccharomyces pombe, and Nhalp from Saccharomyces cerevisiae. The open reading frames (ORFs) from ZSOD2 or ZSOD22 were inserted into a yeast expression vector pYES2, and their constructs (pZSOD2 and pZSOD22) were used to transform the salt-sensitive S. cerevisiae. pZSOD2- or pZSOD22-harboring-recombinant S. cerevisiae cells showed increases in salt tolerance. However, the Z. rouxii disruptant of ZSOD22 did not show any phenotypes related to salt tolerance or osmotolerance, unlike that of ZSOD2. The transcriptional expression of ZSOD22 was not observed by Northern blot analysis even in Z. rouxii cells subjected to NaCl-shock. From these results we conclude that although Z. rouxii includes at least two copies of the Na+/H(+)-antiporter gene (ZSOD2 and ZSOD22), ZSOD2 encodes a functional product as an antiporter and ZSOD22 is poorly transcribed, if at all.

Amino Acid Sequence↗