Biomedical subjects
Publications and source records attributed to Y Wada.
Paramagnetic shifts have been observed for the first time in rare-earth zeolites; the 29Si MAS NMR spectra of rare-earth ion-exchanged low silica X show a large range of isotropic chemical shifts that can be attributed to Fermi contact interactions with the lanthanide electronic moments.
Subunit gamma of the ATP synthase F(1) sector is located at the center of the alpha(3)beta(3) hexamer and rotates unidirectionally during ATP hydrolysis, generating the rotational torque of approximately 45 pN.nm. A mutant F(1) with the betaSer-174 to Phe substitution (betaS174F) in the beta subunit generated lower torque ( approximately 17 pN.nm), indicating that betaS174F is mechanically defective, the first such mutant reported. The defective rotation of betaS174F was suppressed by a second-site mutation, betaGly-149 to Ala, betaIle-163 to Ala, or betaIle-166 to Ala in the same subunit, but not by betaLeu-238 to Ala. These results suggest that the region between betaGly-149 and betaSer-174 plays an important role in the coupling between ATP hydrolysis and mechanical work.
The 23-kDa proteolipid subunit of mouse vacuolar-type proton-translocating ATPase (V-ATPase) was predicted to be a hydrophobic polypeptide of 205 amino acid residues with five putative transmembrane segments. It exhibits sequence similarity to Vma16p of Saccharomyces cerevisiae and vha-4 of Caenorhabdittis elegans (83 and 84%, respectively). Southern blot analysis indicated that the proteolipid is encoded by a single gene, Atp6f, in the mouse genome. Atp6f was mapped to approximately 55 cM on chromosome 4, and its genomic organization is similar to that of the human gene: 8 exons separated by 7 introns, with boundaries matching the GT-AG rule. RNA blotting demonstrated that Atp6f is transcribed as 1.0- and 1.8-kb mRNAs in multiple tissues to varying degrees. The major transcription initiation sites are at -13 and -58 bp upstream of the translation initiation codon. The epitope-tagged 23-kDa protoelipid was localized in endomembrane organelles in CHO cells, as expected for a component of a vacuolar-type proton pump.
Prostate cancer has a high propensity to metastasize to bone, which often resists hormone, radiation, and chemotherapies. Because of the reciprocal nature of the prostate cancer and bone stroma interaction, we designed a cotargeting strategy using a conditional replication-competent adenovirus to target the growth of tumor cells and their associated osteoblasts. The recombinant Ad-OC-E1a was constructed using a noncollagenous bone matrix protein osteocalcin (OC) promoter to drive the viral early E1a gene with restricted replication in cells that express OC transcriptional activity. Unlike Ad-PSE-E1a, Ad-OC-E1a was highly efficient in inhibiting the growth of PSA-producing (LNCaP, C4-2, and ARCaP) and nonproducing (PC-3 and DU145) human prostate cancer cell lines. This virus was also found to effectively inhibit the growth of human osteoblasts and human prostate stromal cells in vitro. Athymic mice bearing s.c. androgen receptor-negative and PSA-negative PC-3 xenografts responded to a single intratumoral administration of 2 x 10(9) plaque-forming unit(s) of Ad-OC-E1a. In SCID/bg mice, intraosseous growth of androgen receptor-positive and PSA-producing C4-2 xenografts responded markedly to i.v. administrations of a single dose of Ad-OC-E1a. One hundred percent of the treated mice responded to this systemic Ad-OC-E1a therapy with a decline of serum PSA to an undetectable level, and 80% of the mice with PSA rebound responded to the second dose of systemic Ad-OC-E1a. Forty percent of the mice were found to be cured by systemic Ad-OC-E1a without subsequent PSA rebound or tumor cells found in the skeleton. This cotargeting strategy shows a broader spectrum and appears to be more effective than systemic Ad-PSE-E1a in preclinical models of human prostate cancer skeletal metastasis.
The vacuolar-type H+ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical alpha- and beta-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.
BACKGROUND: Acute rejection of allografts remains a significant problem in clinical transplantation, and the fundamental mechanism underlying this rejection are as yet only poorly elucidated. Recently, DNA microarrays have come into use for the study of gene expression profiles, and we have taken advantage of this new technology to investigate acute rejection. We compared mRNA profiles in murine cardiac allografts with isografts using DNA microarrays with probe sets corresponding to more than 11,000 mice genes. METHODS: We screened for gene expression changes in murine cardiac allografts between fully incompatible mice strains (BALB/c H2d to C3H/He H2k) using a DNA microarray. The heart was heterotopically transplanted. Allografts (BALB/c to C3H/He) were removed on days 1, 3, and 5. As a control, isografts (C3H/He to C3H/He) harvested on days 1, 3, and 5 and native hearts of both strain mice (C3H/He and BALB/c) were obtained. RESULTS: On day 5, interferon-gamma (IFN-gamma) and many IFN-gamma-inducible genes were profoundly induced in the allograft relative to isograft. Monokine induced by IFN-gamma was most profoundly induced followed by inducibly expressed GTPase and Lmp-2. IFN-gamma was also profoundly induced. The induction was detectable from day 3. In contrast, genes regulated by other cytokines exhibited only modest changes. CONCLUSION: IFN-gamma-inducible genes are specifically up-regulated in murine cardiac allografts, suggesting that signaling mediated by IFN-gamma may play an important role in the late phase of acute rejection in vivo.
Explore the source record for details and available documents.
We have identified four genes (vha-5, vha-6, vha-7, and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans, the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43-60% identity and 62-84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem. 273, 6717-6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells. Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4, and vha-11) (Oka, T., and Futai, M. (2000) J. Biol. Chem. 275, 29556-29561). The progenies of worms injected with the vha-5 or vha-6 double-stranded RNA became died at a specific larval stage, whereas the vha-7 double-stranded RNA showed no effect on development. These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner.
A mass screening program for congenital hypothyroidism has markedly improved prognosis of children with congenital hypothyroidism and also revealed several cases with unknown pathogenesis. We here report two independent Japanese multigeneration families with multinodular goiter (MNG) with euthyroidism and with high TSH. The propositi, 3- and 8-year-old girls in two families, were found during a mass screening. An autosomal dominant pattern of inheritance was suggested in both families. The clinical examinations suggested impaired hormonogenesis but discarded known defects in iodine transport, organification, deficiency of hydrogen peroxide, and thyroid peroxidase. Linkage analysis of the two families including 10 members each using 343 microsatellite markers mapped a single locus independently at D3S1618 (theta = 0) on 3q26.1-q26.3 with a two-point LOD score 3.62 (1.81 for each family) and multipoint LOD score of 3.61 (1.80 for each family). Haplotype inspection delimited an 18-cM interval between D3S1565 and D3S3686.
Hartnup disorder is an autosomal recessive phenotype involving a transporter for monoamino-monocarboxylic acids. Genetic analysis of the mouse model mapped its locus to human chromosome 11q13 (8). We report here the results of linkage analysis in two Japanese first cousin-marriage families. In the first family, the proband had Hartnup disorder and his deceased older brother was reported to have had typical Hartnup symptoms. The younger brother of the proband was shown to have decreased tryptophan absorption by oral loading test. In the second family, a 6-year-old girl, the proband, had specific hyperaminoaciduria. DNA was isolated from either blood samples or umbilical cord stumps. Genome-wide screening by homozygosity mapping was conducted. Taking into account that the older brother was affected and the younger brother was a carrier in the first family, homozygosity mapping (LOD score = 3.55) and GENEHUNTER (LOD score = 3.28) locates the locus of the Hartnup disorder on 5p15.
Patients with inborn errors of metabolism undergo liver transplantation, but the effect of transplanting the liver of healthy carriers of these conditions has not been fully clarified. A 6-year-old girl with classical citrullinemia, who repeatedly suffered from hyperammonemia, underwent living-related liver transplantation by using a segment of the liver of her mother, who was a heterozygote carrier for classical citrullinemia. Hyperammonemia alleviated in the patient after the transplantation, thereby dramatically improving her quality of life. Although the levels of plasma and urinary citrulline remained high postoperatively, there was no marked difference in the level of plasma citrulline up to 1 month after surgery when compared with that of previously reported orthotopic liver transplantation cases with classical citrullinemia.
Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.
The expression of human brain-derived neurotrophic factor (BDNF) was investigated in 16 primary human neuroblastomas with favorable biologies, 15 with unfavorable biologies, and in human neuroblastoma cell lines. We demonstrated higher expressions of human BDNF mRNA in neuroblastomas with unfavorable biologies and with N-myc amplification than in those with favorable biologies. For the first time we revealed the composition of splice variants of human BDNF mRNA and analyzed their expression in neuroblastomas by reverse transcription polymerase chain reaction (RT-PCR). Interestingly, human BDNF mRNA consisted of at least six isoforms, four isoforms resembling those of rat BDNF mRNA, a human-specific isoform and a new isoform. The expression of four isoforms were more prominent in tumors with unfavorable biologies than in those with favorable biologies (P<0.05). As previously we had reported, over 80% of the primary tumors expressed either the full-length form of BDNF receptor, TRKB, or a truncated form of TRKB lacking the tyrosine kinase domain. The full-length TRKB was predominantly detected in tumors with unfavorable biologies, and the truncated one in those with favorable biologies. These results suggest that an autocrine and/or paracrine mechanism involving BDNF may stimulate signal transduction via TRKB receptors rich in neuroblastomas with unfavorable biologies, resulting in an aberrant survival of tumor cells.
ATP synthase (F0F1) transforms an electrochemical proton gradient into chemical energy (ATP) through the rotation of a subunit assembly. It has been suggested that a complex of the gamma subunit and c ring (c(10-14)) of F0F1 could rotate together during ATP hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T mutation (c subunit cIle-28 replaced by Thr) was less sensitive to venturicidin than that of the wild type, consistent with the antibiotic effect on the cI28T mutant and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M., and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1 to see the alpha(3)beta(3) hexamer rotation; a biotin tag was introduced into the alpha or beta subunit, and a His tag was introduced into the c subunit. The engineered enzymes could be purified by metal affinity chromatography and density gradient centrifugation. They were immobilized on a glass surface through the c subunit, and an actin filament was connected to the alpha or beta subunit. The filament rotated upon the addition of ATP and generated essentially the same frictional torque as one connected to the c ring. These results indicate that the gammaepsilonc(10-14) complex is a mechanical unit of the enzyme and that it can be used as a rotor or a stator experimentally, depending on the subunit immobilized.
[figure: see text] Various phosphine oxides are efficiently reduced by the use of a methylation reagent and lithium aluminum hydride. Optically active P-chirogenic phosphine oxides are also reduced with inversion of configuration at phosphorus atom by treatment with methyl triflate, followed by reaction with LiAlH4.
A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3'-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing beta-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Gal beta 1-3GalNAc alpha-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO(3)-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1- 4Glc-PA by two-dimensional (1)H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Gal beta 1-3GlcNAc-R) and type 2 (Gal beta 1-4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel beta-Gal-3'-sulfotransferase gene family.
ZnS nanocrystallites (nc-ZnS) prepared in N,N-dimethylformamide (DMF) photocatalyze dehalogenation of halogenated benzenes to benzene as the final product from chlorinated benzenes and to difluorobenzenes from fluorinated benzenes in the presence of triethylamine (TEA) as an electron donor under UV light irradiation (lambda > 300 nm). When CdS nanocrystallites (nc-CdS) are used as a photocatalyst (lambda > 400 nm), halogenated benzenes are photoreductively dehalogenated, yielding trichlorobenzene from hexachlorobenzene and tetrafluorobenzene isomers from hexafluorobenzene as the final products. Photoformed electrons on nc-ZnS and nc-CdS have such negative reduction potentials that these electrons reduce polyhalogenated benzenes, leading to the successive dehalogenation. nc-ZnS exhibits higher photocatalytic activitythan nc-CdS due to the more negative potential of the electrons on nc-ZnS than that on nc-CdS. The higher activities of nc-ZnS and nc-CdS compared to their bulk forms are explained as being due to their quantum size effects and the adsorptive interaction between the substrates and the nanosized photocatalysts.