Biomedical subjects

H Watanabe

Publications and source records attributed to H Watanabe.

At least 541 records · Page 30Linked to original sources

PubMed1999

Dynamic evolution of genomes and the concept of genome space.

A new era in the elucidation of genome evolution has been heralded with the availability of numerous genome sequences. With these data, it has been possible to study evolutionary processes at a greater level of detail in order to characterize features such as gene shuffling, genome rearrangements, base bias composition, and horizontal gene transfer. In this paper, we discuss the evolutionary implications of significant rearrangements within genomes as well as characteristic genomic regions that have been conserved across genomes. This is based on our analysis of orthologous and paralogous genes. We argue that genome plasticity has most likely contributed substantially to the dynamic evolution of genomes. We also describe the characteristic mosaic features of an archaea genome that is comprised of both bacterial and eukaryal elements. Here we investigate base compositional differences as well as the similarity of this species' genes to either bacteria or eukarya. We conclude that these features can be largely explained by the mechanism of horizontal gene transfer. Finally, we introduce the concept of genome space which is defined as the entire set of genomes of all living organisms. We explain its usefulness to describe as well as to gain deeper insight into the general features of the dynamic genomic evolutionary process.

Archaea↗

PubMed1999

Expansion of extrathymic T cells as well as granulocytes in the liver and other organs of granulocyte-colony stimulating factor transgenic mice: why they lost the ability of hybrid resistance.

When we attempted to characterize the immunological state in G-CSF transgenic mice, a large number of not only granulocytes but also lymphoid cells expanded in various immune organs. Such lymphoid cells were present at unusual sites of these organs, e.g., the parenchymal space in the liver. We then determined the phenotype of these lymphoid cells by immunofluorescence tests. It was demonstrated that CD3intIL-2Rbeta+ cells (i.e., extrathymic T cells), including the NK1.1+ subset of CD3int cells (i.e., NKT cells), increased in the liver and all other tested organs. These T cells as well as NK cells mediated NK and NK-like cytotoxicity, especially at youth. However, they were not able to mediate such cytotoxicity in the presence of granulocytes. This result might be associated with deficiency in the hybrid resistance previously ascribed to these mice. In other words, G-CSF transgenic mice had a large number of extrathymic T cells (including NKT cells) and NK cells that mediate hybrid resistance, but their function was suppressed by activated granulocytes. Indeed, these granulocytes showed an elevated level of Ca2+ influx upon stimulation. The present results suggest that, in parallel with overactivation of granulocytes, extrathymic T cells and NK cells are concomitantly activated in number but that their function is suppressed in G-CSF transgenic mice.

Age Factors↗

PubMed1999

14-3-3 protein eta chain gene (YWHAH) polymorphism and its genetic association with schizophrenia.

Recent genetic analyses have suggested a linkage between schizophrenia and the chromosomal region 22q12-q13. 14-3-3 protein, abundant in the brain, mediates interactions between diverse molecules of biological activities; its gene was recently mapped to chromosome 22q12.1-q13.1. We therefore investigated allele frequencies of a variable number of tandem repeat (VNTR) in the 5'-noncoding region of the 14-3-3 eta chain gene in controls and schizophrenics. The frequencies of the two-repeat allele were significantly higher (P < 0.05) in the schizophrenics, and particularly in those with onset before age 22 (early-onset schizophrenics, P < 0.02), than in the controls. The odds ratio was significantly increased in the early-onset schizophrenics homozygous for the two-repeat allele (OR = 3.3, 95% CI = 1.1-9.7). The 14-3-3 eta chain gene is a potential susceptibility gene for schizophrenia, and particularly for early-onset schizophrenia.

14-3-3 Proteins↗

PubMed1999

Co-localization of mitochondrial and double minute DNA in the nuclei of HL-60 cells but not normal cells.

In an attempt to isolate genes located on double minute (Dmin) DNA in HL-60 cells, we prepared DNA probe from purified micronuclei. Micronucleation was induced in HL-60 cells by treatment with hydroxyurea. Screening of a cDNA library unexpectedly produced a number of clones containing mitochondrial DNA (mtDNA) sequences. Here, we show that amplified mtDNA sequences were localized in nuclei and micronuclei of HL-60 and COLO 320DM cells, but not in nuclei of WI-38 normal human fibroblasts or peripheral blood T-cells. To unequivocally demonstrate the presence of mtDNA inside of nuclei and micronuclei, we obtained tomographic fluorescence in situ hybridization (FISH) images of mtDNA by confocal microscopy of consecutive sections of paraformaldehyde (PFA)-fixed material. We also located mtDNA in nuclear buds and purified micronuclei. Dmin DNA and mtDNA were always located at similar sites. The mechanisms of nuclear retention of mtDNA and Dmin DNA and the resulting influence on tumorigenesis are discussed.

Cell Cycle↗

PubMed1999

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

Adenylyl Cyclase Inhibitors↗

PubMed1999

Abundance of NKT cells in the salivary glands but absence thereof in the liver and thymus of aly/aly mice with Sjögren syndrome.

It is known that ALY/Nsc Jcl-aly/aly (aly/aly) mice that congenitally lack lymph nodes fall victim to Sjögren syndrome as a function of age. We investigated how TCRint cells of extrathymic origin and TCRhigh cells of thymic origin are distributed in various organs of these mice. Although the distribution of T-cell subsets was not different between control aly/+ and aly/aly mice in youth in any of the tested organs, the proportion of TCRint cells in the liver and spleen of aly/aly mice increased with aging. Usually, TCRint cells in the liver comprise a half-and-half mixture of a NK1. 1(+) subset (i.e., NKT cells) and a NK1.1(-) subset. In constrast, almost all expanding TCRint cells in various immune organs of aly/aly mice were found to be NK1.1(-). A large proportion of lymphocytes, including NK cells and TCRint cells, were also present in the salivary glands of aly/aly mice. Interestingly, these TCRint cells in the salivary glands contained an NK1.1(+) subset (i.e., NKT cells) that used an invariant chain of Valpha14Jalpha281 for TCRalphabeta (>50%). Moreover, gammadeltaT cells that used Vgamma 1, 2, 4/Vdelta 1, 4, 6 mRNAs, different from those of gammadeltaT cells in the liver and intestine, were abundant. Possibly reflecting the in situ generation of these T cells in the salivary glands, the expression of RAG-2 mRNA was evident by the RT-RCR method. These results suggest that (i) inflammatory lymphocytes that evoke Sjögren syndrome in aly/aly mice are NK cells or TCRint cells (both NK1.1(+) and NK1.1(-) subsets) and (ii) TCRint cells in the salivary glands might be generated in situ.

Animals↗

PubMed1999

Interaction of the small G protein RhoA with the C terminus of human phospholipase D1.

Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biological settings including the regulation of membrane vesicular trafficking. PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha). This stimulation has been proposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two-hybrid system to attempt to identify these sites. Successful interaction of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of the CAAX box from RhoAVal-14 decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1. The specificity of the interaction was validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation. These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.

Binding Sites↗

PubMed1999

Poly(L-lysine)-graft-dextran copolymer promotes pyrimidine motif triplex DNA formation at physiological pH. Thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DNA↗

PubMed1999

Mutations in the p53 and scid genes do not cooperate in lymphomagenesis in doubly heterozygous mice.

Analysis of double mutant mice of the p53 and scid genes, which have a combination of cell cycle checkpoint/apoptosis and DNA repair defects, shows that the latter defect synergistically enhances lymphoma development with loss of the former function. These mice lack the ability to eliminate lymphocytes predisposed to neoplastic transformation resulting from faulty antigen receptor gene rearrangement. Here we examine the cooperativity in double heterozygotes of p53 and scid in which normal development of lymphocytes is not impaired. MSM mice carrying a p53-knockout allele were crossed with BALB/c mice heterozygous for the scid locus and 129 offspring were obtained. They were subjected to gamma-ray irradiation, 84 thymic lymphomas being generated. The tumors and host mice were genotyped of p53 and scid. Among 42 mice developing p53-deficient lymphomas, scid/+ and +/+ genotypes did not provide difference in onset and latency. Besides, allelic loss of the Scid gene occurred at a high frequency in those lymphomas but the loss exhibited no allelic bias. The results suggest that the scid/+ genotype is not a modifier of loss of p53 function in the double heterozygotes.

Alleles↗

PubMed1999

Different effects of unilateral and bilateral hippocampal lesions in rats on the performance of radial maze and odor-paired associate tasks.

The hippocampus plays an important role in the declarative or explicit memory in humans and is necessary for allocentric spatial learning and olfactory memory in animals. In primates and rodents, the bilateral hemispheres of the brain (especially the forebrain) symmetrically and asymmetrically contribute to diverse cognitive manipulations. In this study, we investigated the role of the hippocampus in spatial memory and in odor-paired associate memory by unilaterally or bilaterally lesioning this region in rats. The bilateral removal, but not the unilateral removal, of the hippocampus impaired both the acquisition of spatial working memory in the radial maze task and the retrieval of maze performance tested 1 month after the acquisition trials. In contrast, neither bilateral nor unilateral removal impaired the odor-paired associate learning. These findings suggest that the hippocampus is critical to the spatial memory, and that a unilateral hippocampus is sufficient for executing a spatial task. The present results also indicate that the hippocampus plays a minor role in odor-dominated associate learning and that some kinds of memories in rats may be processed independently by the left or right hippocampus.

Animals↗

PubMed1999

Quantitative DNA analysis of low-level hepatitis B viremia in two patients with serologically negative chronic hepatitis B.

Low-level viremia due to hepatitis B virus (HBV) was demonstrated in the sera of two patients diagnosed previously as having non-B, non-C chronic hepatitis. Both patients had a "silent" HBV infection, because they were negative for both hepatitis B surface antigen (HBsAg) and anti-hepatitis B core antibody. The TaqMan chemistry polymerase chain reaction (PCR) amplified the HBV DNA, enabling quantitation of the virus in their sera. Their serum HBV DNA concentrations were low: the amount of each HBV S or X gene amplified showed there were approximately 10(3) copies/ml and HBV DNA was detected occasionally during clinical follow-up. Positive HBsAg staining in liver tissues was demonstrated by an immunoperoxidase technique. Vertical transmission of silent HBV from one patient to her daughter was confirmed. Direct nucleotide sequencing of the amplified HBV X region revealed several mutations, suggesting reduced viral replication. One patient had a T-to-C mutation at the extreme 5'-terminus of the direct repeat 2 region and the other exhibited a coexisting X region with a 155-nucleotide deletion. These findings suggest that HBV replication is suppressed considerably in patients with silent hepatitis B.

Adult↗

PubMed1999

No prognostic significance of p53 expression in esophageal squamous cell carcinoma.

BACKGROUND AND OBJECTIVES: It is generally accepted that the overexpression of p53 protein is associated with poor prognosis in breast, colorectal, and other types of cancer. However, the prognostic significance of p53 aberrations in esophageal squamous cell carcinoma has yet to be determined. We attempted to analyze the relationship between p53 expression and the clinicopathologic features of esophageal squamous cell carcinoma by reviewing the medical records of a large patient population. Our study of esophageal squamous cell carcinoma involves the largest patient population to date. METHODS: p53 expression in formalin-fixed, paraffin-embedded samples of 239 patients with primary esophageal squamous cell carcinoma (TNM stage I:79 cases, stage II: 82 cases, stage III: 78 cases), who underwent esophageal resection without additional treatment, were analyzed by immunohistochemical staining using a polyclonal antibody, RSP53. The relationships between p53 immunoreactivity and prognostic factors were determined by the chi(2) test, and the prognostic impact of p53 protein expression was analyzed by univariate and multivariate survival analyses. RESULTS: In 115 (48.1%) of 239 esophageal tumors, nuclear immunoreactivity for the p53 protein was detected. The expression of the p53 protein did not correlate with sex, age, histological grading, lymph node metastasis, vascular invasion, or TNM stage. Similarly, p53 expression did not correlate with prognosis in univariate and multivariate survival analysis. CONCLUSIONS: The expression of the p53 gene product had no impact on the prognosis of esophageal squamous cell carcinoma.

Adult↗

PubMed1999

The common 18-base pair deletion at codons 418-423 of the E-cadherin gene in differentiated-type adenocarcinomas and intramucosal precancerous lesions of the stomach with the features of gastric foveolar epithelium.

Alterations of the E (epithelial)-cadherin gene are often involved in undifferentiated-type carcinomas of the stomach, but not in the differentiated type. Recently, it was recognized that certain differentiated-type carcinomas possess the features of gastric foveolar epithelium. Such carcinomas are closely linked with undifferentiated-type carcinomas. Nineteen lesions (ten differentiated-type carcinomas and nine precancerous lesions) with a predominant gastric foveolar phenotype, as determined not only by mucin histochemistry and immunohistochemistry but also by morphology, were analysed for mutations of the E-cadherin gene using the nested polymerase chain reaction single-strand conformation polymorphism method. E-cadherin gene mutation was detected in 4 of 19 lesions (21 per cent) (three precancerous and one cancerous). Sequencing revealed an 18 bp deletion corresponding to codons 418-423 of exon 9 common to all four lesions. This mutation was previously reported in an advanced carcinoma of undifferentiated type. The results suggest that these tumours progress to undifferentiated-type carcinomas through loss of E-cadherin function.

Adenocarcinoma↗

PubMed1999

Voltage-induced variation of distribution coefficient in electrochromatography.

Upon application of an electric field along a column packed with anion exchange support materials, the output of a recorder jumps beyond the baseline, and with continued application a peak is obtained. If, instead, the electric field is applied in a pulsed manner, two peaks result. These observations are explained as caused by a change in the concentration of the solvent. Applying positive and negative voltages produces positive and negative peaks, respectively. The peak height depends on the value of applied voltage. Simulations using the plate height theory agree well with chromatograms obtained experimentally. The value of the local applied voltage is also estimated. Overall, the behavior is something like an injection arising from the pulsed application of an electric field. We suggest that voltage pulsing might be used as a new technique for sample injection.

Chromatography, Liquid↗

PubMed1999

Preincubation of recombinant Ipa proteins of Shigella sonnei promotes entry of non-invasive Escherichia coli into HeLa cells.

Invasion plasmid antigens of Shigella sonnei, IpaB, C, D, were expressed as fusion proteins either with maltose-binding protein (MBP) or Strept-tag sequence. Affinity-purified IpaB and IpaD were separated from MBP by digestion with Factor Xa. Recombinant IpaC having Strept-tag sequence at its C-terminal was also purified by avidin affinity column chromatography. These recombinant proteins showed the ability to cause non-invasive Escherichia coli K-12 to internalize HeLa cell only when all of the proteins were preincubated with the bacterial prior to the inoculation of the mixture into HeLa cell culture. Electron microscopy also showed internalized bacteria within HeLa cells suggesting that functional complex of invasins (IpaB, C and D) were formed in vitro.

Antigens, Bacterial↗

PubMed1999

Yersinia pseudotuberculosis blocks the phagosomal acidification of B10.A mouse macrophages through the inhibition of vacuolar H(+)-ATPase activity.

Yersinia pseudotuberculosis survived and multiplied in the phagosomes of B10.A mouse peritoneal macrophages. As one of the possible mechanisms for the bacteria's survival in the phagosomes, we demonstrated that live Y. pseudotuberculosis inhibited the phagosomal acidification; pH within phagosomes containing the live Y. pseudotuberculosis remained at about 6.0, whereas pH within phagosomes containing the dead Y. pseudotuberculosis fell to about 4. 5. This ability to inhibit intraphagosomal acidification was also shared by mutants lacking the 42 Md virulence plasmid, indicating that it is chromosomally encoded. The phagosomes containing dead bacteria raised the pH to 6.2 after the treatment of their macrophages with an inhibitor (bafilomycin A1) specific for V-ATPase. Although the amount of V-ATPase in the A and B subunits on the phagosomes was not significantly different between the live and dead bacteria infection, the phagosomes containing live bacteria had a 10-fold smaller V-ATPase activity than those containing the dead bacteria. These results indicated that the inhibition of phagosomal acidification by Y. pseudotuberculosis infection was due to the attenuation of V-ATPase activity, and not due to the exclusion of V-ATPase subunits from the phagosome membrane as found in Mycobacterium avium.

Animals↗

PubMed1999

Validity of the right ventricular Doppler index for assessment of severity of congestive heart failure in patients with dilated cardiomyopathy.

This study was designed to clarify the validity of Doppler indices for assessment of the severity of congestive heart failure in patients with dilated cardiomyopathy. Left ventricular (LV) and right ventricular (RV) Doppler indices, defined as the sum of isovolumic contraction time and relaxation time divided by the ejection time, were obtained in 30 normal subjects and 35 patients with ischemic or idiopathic dilated cardiomyopathy. We analyzed the correlation between New York Heart Association (NYHA) functional class, right-sided cardiac pressure, and the Doppler indices. Patients with dilated cardiomyopathy revealed significantly higher LV and RV Doppler indices than normal subjects; however, the LV Doppler index did not correlate significantly with NYHA functional class or right-sided cardiac pressure. On the other hand, the RV Doppler index correlated significantly with NYHA functional class (rs = 0.83, P < 0.001), pulmonary capillary wedge pressure (r = 0.80, P < 0.001), pulmonary artery systolic pressure (r = 0.81, P < 0.001) and pulmonary artery diastolic pressure (r = 0.77, P < 0.001). Both RV isovolumic contraction and relaxation time also correlated significantly with right-sided cardiac pressure. In 12 patients who improved from NYHA III-IV to I-II after treatment, the RV Doppler index decreased from 0.84 +/- 0.25 to 0.33 +/- 0.10 (P < 0.001), while the LV Doppler index did not change significantly (0.72 +/- 0.27 to 0.68 +/- 0.23, P = 0.53). These results suggest that the RV Doppler index is useful for assessment of the severity of congestive heart failure in patients with dilated cardiomyopathy.

Cardiac Catheterization↗

PubMed1999

Changes in pulsed-field gel electrophoresis patterns in clinical isolates of enterohemorrhagic Escherichia coli O157:H7 associated with loss of Shiga toxin genes.

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains showed disappearance of a 70- or 80-kb fragment in their patterns associated with loss of Shiga toxin genes during maintenance or subcultivation. Hybridization experiments with a DNA probe complementary to Shiga toxin sequences revealed that the Shiga toxin genes in the parental strain were located on fragments the same size as the lost fragments from the toxin-negative derivatives. The evidence indicates that PFGE pattern of EHEC O157:H7 may change due to loss of Shiga toxin genes, which is likely to be associated with curing of Shiga toxin gene carrying phages in vitro.

Bacterial Toxins↗