Biomedical subjects

Y Wada

Publications and source records attributed to Y Wada.

At least 325 records · Page 18Linked to original sources

PubMed1998

Identification of the copper chaperone, CUC-1, in Caenorhabditis elegans: tissue specific co-expression with the copper transporting ATPase, CUA-1.

A cDNA encoding a putative copper chaperone protein, CUC-1, was cloned from Caenorhabditis elegans. CUC-1 had the characteristic motifs of MTCXXC and KKTGK, and showed 49.3 and 39.1% sequence identity with yeast Atx1p and human HAH1, respectively. Expression of CUC-1 cDNA complemented a null atx1 mutant, the yeast copper chaperone gene, thus demonstrating that CUC-1 is a functional copper chaperone. Studies with transgenic worms indicated that cuc-1 and cua-1, which encodes the copper transporting ATPase, are expressed together in intestinal cells of adult and hypodermal cells in the larvae. cua-1 was also expressed in pharyngeal muscle but cuc-1 was not. These results suggest that CUC-1 and CUA-1 constitute a copper trafficking pathway similar to the yeast counterparts in intestinal and hypodermal cells, and CUA-1 may have a different function in pharyngeal muscle.

Adenosine Triphosphatases↗

PubMed1998

Stability of the Escherichia coli ATP synthase F0F1 complex is dependent on interactions between gamma Gln-269 and the beta subunit loop beta Asp-301-beta Asp-305.

The role of the conserved sequence motif 301DDLTDP306 in the F0F1 ATP synthase beta subunit was assessed by mutagenic analysis in the Escherichia coli enzyme. Mutations gave variable effects on F1 sector activity, stability, and membrane binding to the F0 sector. Upon solubilization, F1 sectors of the betaD302E and betaD305E mutants (betaAsp-302 and betaAsp-305 replaced by glutamate) dissociated into subunits, while mutants with other beta305 substitutions failed to assemble. Membrane ATPase activities of beta301 and 302 mutants were 20-70% of wild type. Replacements of the gamma subunit Gln-269 had similar effects. The membrane ATPase activities of the gammaQ269E or gammaQ269D mutants were significantly lower and their F1 sectors dissociated into subunits upon solubilization. These results suggest that the beta301-305 loop and the gamma subunit region around Gln-269 form a key region for the assembly of alpha3 beta3 gamma complex. These results are consistent with the X-ray crystallographic structure of bovine F1 (J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1994) Nature 370, 621-628) where the beta301DDLTD305 loop directly interacts with gammaGln-269.

Amino Acid Substitution↗

PubMed1998

Neurotrophic action of lipocortin 1 derived from astrocytes on cultured rat cortical neurons.

The lipocortins are a family of structurally related proteins, namely an annexin family, that exerts a variety of cellular functions through Ca2+-dependent binding to phospholipase A2 [EC 3.1. 1.4], including a crucial role in the central nervous system (CNS) such as antipyrogenic, thermoregulatory and neuroprotective agents in vivo. To elucidate the paradigm of lipocortin 1 functions in the CNS, we have first demonstrated (1) the induction and subsequent extracellular secretion of LC1 by glucocorticoid in cultured rat astrocytes, and (2) neurotrophic activities (survival-promoting, neuritogenic and synaptogenic actions on rat cortical neurons) of recombinant LC1. Time-and dose-dependent experiments of a synthetic glucocorticoid, dexamethasone (DEX), on rat cortical astrocytes in culture revealed that the expression of the intracellular LC1 mRNA and protein were significantly augmented by DEX (1 microM). In addition, DEX evoked an extracellular secretion of LC1 without its cytotoxic effects. Furthermore, the recombinant LC1 appeared to promote not only the survival and neurite outgrowth but also the synaptogenesis of embryonal rat cortical neurons. These results suggest that LC1 induced and selectively released from astrocytes by either endogenously or exogenously introduced glucocorticoids may play a specific and essential role on development and regeneration of the central nervous system.

Animals↗

PubMed1998

PCR on cerebrospinal fluid to show influenza-associated acute encephalopathy or encephalitis.

BACKGROUND: Except for Reye's syndrome, influenza-associated acute encephalopathy or encephalitis is not universally recognised. We did a multicentre study of laboratory and clinical data for patients with influenza-associated acute encephalopathy or encephalitis. METHODS: In Nagoya, Japan, ten patients with acute encephalopathy or encephalitis associated with influenza-like illness were admitted to our hospitals between April, 1996, and March, 1997. We collected clinical, laboratory and serological data and assessed cerebrospinal fluid samples by PCR for influenza A and B. FINDINGS: Seven patients, aged 22 months to 4 years, had evidence of recent influenza infection, six with type-A/Hong Kong (H3N2) and one with type B. The first sign in the central nervous system appeared within 2 days of fever in all but one patient. The first sign of involvement of the central nervous system was generalised convulsions in all patients. Two patients died, one had sequelae, and four survived without sequelae. PCR for influenza type A was positive for five patients. INTERPRETATION: The results of PCR suggest that at least part of the influenza type A genome existed in the central nervous system. Influenza-associated acute encephalopathy or encephalitis in young children deserves wider recognition.

Acute Disease↗

PubMed1998

Cancer-associated expression of glycolipid sulfotransferase gene in human renal cell carcinoma cells.

Human renal cell carcinoma (RCC) tissue and a cell line derived therefrom, SMKT-R3, showed markedly increased glycolipid sulfotransferase [cerebroside sulfotransferase (CST); EC 2.8.2.11] activity and accumulated sulfoglycolipids. Recently, we cloned a human CST cDNA from a SMKT-R3 cDNA library (K. Honke et al., J. Biol. Chem., 272: 4864-4868, 1997). In this study, we investigated the expression of the CST gene in seven human RCC lines (SMKT-R1, SMKT-R2, SMKT-R3, SMKT-R4, TOS-1, TOS-2, and ACHN) and their normal counterpart, human renal proximal tubular cells. On Northern blot analysis, a marked increase of CST mRNA was observed in every RCC line, except for ACHN, as compared with normal cells. ACHN cells showed a slightly increased level of CST mRNA. CST activity was correlated with the amount of mRNA. Sulfoglycolipid analysis revealed that expression of lactosylceramide sulfate was correlated with the CST level. Furthermore, we examined the effects of epidermal growth factor (EGF), tetradecanoylphorbol-13-acetate, and genistein, which are known to regulate CST activity in SMKT-R3 cells, on CST-gene expression in various RCC cells. On treatment with EGF, CST mRNA time-dependently increased in accord with its activity in SMKT-R3 cells. Yet, augmentation by EGF was only observed in SMKT-R3. In contrast, a reduction of CST mRNA and activity by tetradecanoylphorbol-13-acetate and genistein was observed in all of the lines examined. Taken together, these findings indicate that in human RCC cells, the CST gene is generally overexpressed via a signaling pathway involving protein kinase-C and tyrosine kinases.

Carcinoma, Renal Cell↗

PubMed1998

Population and family studies of dihydropyrimidinuria: prevalence, inheritance mode, and risk of fluorouracil toxicity.

To evaluate the prevalence of dihydropyrimidinuria (DHPuria), we analyzed urine samples from 21,200 healthy Japanese infants, and found two cases of DHPuria without clinical symptoms. Based on this result, we estimated the prevalence to be approximately 1/10,000 births in Japan. In addition, we analyzed pyrimidine catabolism on a previously reported family with an adult DHPuria case. We newly identified the sister of the propositus as the second case of DHPuria in this family, because she excreted large amounts of dihydrouracil and dihydrothymine. The parents and the child of the propositus showed slight increases of dihydrouracil and dihydrothymine. This is the first family with 2 cases of DHPuria, indicating that DHPuria is an inherited condition. To determine the inheritance of DHPuria in this family and to examine the risk of 5-fluorouracil (5-FU) toxicity, a uracil loading test was performed on the parents. Urinary dihydrouracil concentrations in the parents after the loading were several times higher than those in normal control persons, the finding being consistent with DHPuria heterozygotes. This, along with data on the propositus, his sister, and his child, indicates that DHPuria is an autosomal recessive condition. In addition, DHPuria homozygotes may have a high risk of 5-FU toxicity, while the risk is relatively low in heterozygotes.

Adult↗

PubMed1998

Effects of glucose and insulin on rat apolipoprotein A-I gene expression.

We have examined the regulation of apolipoprotein A-I (apoA-I) gene expression in response to glucose and insulin. In Hep G2 cells, endogenous apoA-I mRNA was suppressed by one-half or induced 2-fold following 48 h of exposure to high concentrations of glucose (22.4 mM) or insulin (100 microunits/ml), respectively, compared with control. Transcriptional activity of the rat apoA-I promoter (-474 to -7) in Hep G2 cells paralleled endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin. Deletional analysis showed that a 50-base pair fragment spanning -425 to -376 of the promoter mediated the effects of both insulin and glucose. Within this DNA fragment there is a motif (-411 to -404) that is homologous to a previously identified insulin response core element (IRCE). Mutation of this motif abolished not only the induction of the promoter by insulin but also abrogated its suppression by glucose. Electrophoretic mobility shift assay analysis of nuclear extracts from Hep G2 cells revealed IRCE binding activity that formed a duplex with radiolabeled probe. The IRCE binding activity correlated with insulin induction of apoA-I expression. In summary, our data show that glucose decreases and insulin increases apoA-I promoter activity. This effect appears to be mediated by a single cis-acting element.

Animals↗

PubMed1998

Localization of the apoptosis-inducing activity of lupus anticoagulant in an annexin V-binding antibody subset.

Lupus anticoagulant (LAC) is associated with arterial and venous thrombosis, thrombocytopenia, and recurrent fetal loss. We have reported previously that plasma with LAC activity induces apoptosis in endothelial cells and binds annexin V (Nakamura, N., Y. Shidara, N. Kawaguchi, C. Azuma, N. Mitsuda, S. Onishi, K. Yamaji, and Y. Wada. 1994. Biochem. Biophys. Res. Commun. 205:1488-1493). In this study, we separated two IgG antibody fractions, one with and one without affinity for annexin V, from 10 patients with LAC. LAC and apoptotic activities were localized in the annexin V-binding fraction in all 10 patients. DNA fragmentation was dose-dependent, paralleling the amount of IgG added to the human umbilical vein endothelial cell culture medium, and was inhibited by preincubation with annexin V. Removal of the antiphospholipid antibodies from patient IgG with phospholipid liposomes did not abolish the apoptosis-inducing activities or binding to annexin V. These results imply that patients with LAC often have antibodies that do not bind phospholipids and are responsible for the induction of apoptosis in endothelial cells.

Adult↗

PubMed1998

Synthesis and structure-activity relationships of carboxyflavones as structurally rigid CysLT1 (LTD4) receptor antagonists.

The synthesis and CysLT1 receptor affinities of a new series of highly rigid 3'- and 4'-(2-quinolinylmethoxy)- or 3'- and 4'-[2-(2-quinolinyl)ethenyl]-substituted, 6-, 7-, or 8-carboxylated flavones are described. CysLT1 receptor affinities of the flavones (down to 11 nM) were determined by their ability to displace [3H]LTD4 from its receptor in guinea pig lung membranes. Structure-affinity relationship studies showed that the relative positions of the carboxylic acid and the quinoline moiety were critical for CysLT1 affinities. While the carboxyl is optimal in the 8 position but tolerated in the 6 position, only the 6- and not the 8-tetrazole has significant activity. The quinoline moiety may be connected to the flavone skeleton by an ethenyl or a methoxy linker, but the substitution position is important for high affinity, especially in the 6-carboxylated flavones. 4'-Substituted 6-carboxyflavones are essentially inactive, whereas the 3'-substituted analogues have submicromolar CysLT1 affinity. Replacement of the quinoline by other heteroaromates generally leads to decreased affinities, with the phenyl and naphthyl analogues displaying only little or no affinity, while the 7-chloroquinoline analogue is comparable in activity to the quinoline. Flavones having CysLT1 receptor affinities of 10-30 nM were selected for determination of their inhibitory effects on the LTD4-induced contraction of guinea pig ileum in vitro. The IC50 values ranged between 15 and 100 nM. Compound 5d (8-carboxy-6-chloro-3'-(2-quinolinylmethoxy)flavone, VUF 5087) was selected for further research because of its high potency in the functional assay. This series contains the most rigid CysLT1 receptor antagonists known to date, and they are useful in the development of a CysLT1 antagonist model, which is discussed in the companion paper.

Animals↗

PubMed1998

Domain- and site-specific phosphorylation of bovine NF-L by Rho-associated kinase.

Rho-associated kinase (Rho-kinase), the putative target of the small GTP-binding protein Rho, phosphorylated neurofilament protein (NF-L) in vitro with approximately 1 mole phosphate per mole NF-L. Phosphorylated NF-L no longer formed the 10 nm filaments, and NF-L filaments were phosphorylated with a result of nearly complete disassembly. NF-L phosphorylated by Rho-kinase was digested with trypsin, and digested fragments were assigned by MALDI/TOF. Unique phosphorylation sites were found at Ser-26 and Ser-57 in the head domain of NF-L. These results indicate that domain- and site-specific phosphorylation by Rho-kinase may regulate the assembly-disassembly of NF-L filaments.

Amino Acid Sequence↗

PubMed1998

Identification of rhodopsin in the pigeon deep brain.

We detected rhodopsin gene expression in the pigeon lateral septum, a photosensitive deep brain region that is responsible for the photoperiodic gonadal response. The nucleotide sequence of the deep brain rhodopsin cDNA clone exactly matched that of the retinal one, indicating that a single rhodopsin gene is transcribed in the two tissues. Immunohistochemical analysis localized rhodopsin in the cerebrospinal fluid-contacting neurons, which have been assumed to be photoreceptive cells in the deep brain. Pigeon rhodopsin seems to play dual important roles in the visual and non-visual systems, the latter of which contributes to the photoperiodic response.

Amino Acid Sequence↗

PubMed1998

Arrestin gene mutations in autosomal recessive retinitis pigmentosa.

OBJECTIVE: To assess the clinical and molecular genetic studies of patients with autosomal recessive retinitis pigmentosa associated with a mutation in the arrestin gene. DESIGN: Results of molecular genetic screening and case reports with DNA analysis and clinical features. SETTING: University medical center. PATIENTS: One hundred twenty anamnestically unrelated patients with autosomal recessive retinitis pigmentosa. METHODS: DNA analysis was performed by single strand conformation polymorphism followed by nucleotide sequencing to search for a mutation in exon 11 of the arrestin gene. Clinical features were characterized by visual acuity slitlamp biomicroscopy, fundus examinations, fluorescein angiography, kinetic visual field testing, and electroretinography. RESULTS: We identified 3 unrelated patients with retinitis pigmentosa associated with a homozygous 1-base-pair deletion mutation in codon 309 of the arrestin gene designated as 1147delA. All 3 patients showed pigmentary retinal degeneration in the midperipheral area with or without macular involvement. Patient 1 had a sibling with Oguchi disease associated with the same mutation. Patient 2 demonstrated pigmentary retinal degeneration associated with a golden-yellow reflex in the peripheral fundus. Patients 1 and 3 showed features of retinitis pigmentosa without the golden-yellow fundus reflex. CONCLUSIONS: Although the arrestin 1147delA has been known as a frequent cause of Oguchi disease, this mutation also may be related to the pathogenesis of autosomal recessive retinitis pigmentosa. This phenomenon may provide evidence of variable expressivity of the mutation in the arrestin gene.

Adult↗

PubMed1998

Separate analysis of complementary strands of restriction enzyme-digested DNA. An application of restriction fragment mass mapping by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) of a restriction endonuclease digest determines the molecular mass of PCR-amplified DNA more easily than measurement of undigested DNA. With this method, a 664 bp region from the FAS gene could be analyzed and a two-nucleotide deletion in the L1CAM gene was detected in a restriction fragment of 105 nucleotides. Furthermore, the analysis of smaller fragments allowed separate detection of single-stranded oligonucleotides comprising individual digested fragments. This mixture analysis of restriction enzyme digests improves the resolution, sensitivity and accuracy of MALDI/TOF-MS of DNA and is thus expected to facilitate its application to genetic diagnosis.

DNA↗

PubMed1998

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5.

We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34-, CD10+, CD19+, and cytoplasmic mu chain (C mu)-negative surface phenotype from human CD34+ cells purified from umbilical cord blood (CB). When 10(3) CD34+ cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34+ population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a C mu phenotype, and a small number of cells had immunoglobulin mu chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34+ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34+ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.

Animals↗

PubMed1998

Clinicopathological study on hepatocellular carcinoma with lymphocytic infiltration.

We examined the clinicopathologic features of 11 surgically resected hepatocellular carcinomas (HCCs) less than 3 cm in diameter with marked inflammatory cell infiltration (LHCCs). In comparison with the other 152 HCCs without such an infiltration (controls), there were no significant differences in male/female ratio, age, serum alpha-fetoprotein levels, and laboratory and imaging findings. All the 11 LHCC cases were hepatitis B surface antigen (HBsAg) negative and hepatitis C virus antibody positive. Among the 152 controls, 116 cases were also HBsAg negative and HCVAb positive and were referred to as HCV-only controls. The clinical features were not significantly different between the LHCC and the HCV-only controls. The LHCC group tended to have higher numbers of lymphocytes and monocytes in pre- and post-operative peripheral blood, but there were no significant group differences. Recurrence rate was 9.1% in the LHCC group, 47.7% in the controls and 47.5% in the HCV-only controls (P < .01). Five-year survival rate was 100% in the LHCC group, 65.1% in the controls and 68.1% in the HCV-only controls (P < .01). Histologically, remarkable inflammatory cell infiltration, mostly lymphocytic, was observed in the cancerous tissue of the LHCC group. Varying degrees of piecemeal necrosis of cancer nests produced by infiltrating lymphocytes were observed in all the 11 cases. Lymph follicle formation was also found in 10 of 11 cases (90.9%). Liver cirrhosis was associated in 6 LHCC cases (54.5%), in 117 control cases (77.0%), and in 91 HCV-only controls (78.4%). Tumor invasion into the portal vein in the vicinity of the tumor was found in 1 LHCC case (9.1%), in 54 controls (35.5%), and in 34 HCV-only controls (29.3%). Immunohistochemically, most of the infiltrating lymphocytes, other than those in the lymph follicle, were identified as T lymphocyte, and CD8+ T lymphocyte was more predominant than CD4+ T lymphocyte. Better prognosis of the LHCC group could attribute to the anti-tumor effect induced by cellular immunity of CD8+ and CD4+ T lymphocytes, and partly by humoral immunity of B cells which formed lymph follicles.

Adult↗

PubMed1998

Surgery for cholecystocholedocholithiasis in a patient with asymptomatic essential thrombocythemia: report of a case.

Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by a remarkable increase in the platelet count and various clinical symptoms. The perioperative management of patients with ET has yet to be determined, especially when there are no clinical symptoms. We report herein the case of a woman with gallstones whose preoperative hematological data showed remarkable thrombocythemia, but her coagulation studies were normal. The Philadelphia chromosome was negative and bone marrow cytology showed a marked increase in megakaryocytes. Surgery was performed under a diagnosis of cholelithiasis with ET. Considering her severe thrombocythemia and obesity, sufficient heparin was administered to prevent deep vein thrombosis; however, this precipitated postoperative bleeding, necessitating a reoperation. A functional abnormality of the patient's platelets was suspected, and the aggregation by adenosine diphosphate was subsequently found to be significantly inhibited. As patients having ET with no symptoms might have depressed platelet aggregability despite remarkable thrombocythemia, when abdominal surgery is performed, prophylactic therapy for deep vein thrombosis should be avoided. Hence, the preoperative aggregation study of platelets might offer useful information about whether postoperative antithrombotic therapy is indicated.

Anticoagulants↗

PubMed1998

A short region from the LEU2 gene of Saccharomyces cerevisiae functions as an ARS in the yeast Saccharomyces exiguus Yp74L-3.

We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3. A DNA fragment functioning as an ARS in S. exiguus, but not in S. cerevisiae, was shown to exist. The ARS activity for S. exiguus was reduced by the 2-microm plasmid origin of S. cerevisiae when both elements coexisted on a single circular plasmid. Analysis of ARS activity with the PCR products from the fragment revealed that the ARS-acting sequence was located in the 3'-terminal area of the transcribed region of the LEU2 gene of S. cerevisiae. It is suggested that the ARS recognition system in S. exiguus is significantly different from that of S. cerevisiae.

DNA Replication↗

PubMed1998

A SINE species from hippopotamus and its distribution among animal species.

Thirty sequences of a short interspersed repetitive element (SINE) were isolated from genomic DNA of Hippopotamus amphibius (hippopotamus). RNA polymerase III split promoter sequence was observed in all of the 30 sequences; and poly(A)-like structure at 3'-end, as well as direct repeat flanking to the repetitive sequence in many of the 30 sequences. A comparison of the consensus sequence of the 30 sequences with sequences in a DNA database (DDBJ/GENBANK/EMBL) revealed 93% homology to the consensus sequence of a whale SINE, CHR-2, and 73% homology to mouse glutamic acid tRNA. Phylogenetic analysis of tRNA-related regions of the sequences with all of the mouse tRNAs revealed that glutamic acid tRNA was genetically closest to the hippopotamus SINE. In addition, the tRNA-related region of the consensus sequence was folded into a cloverleaf structure as with mouse glutamic acid tRNA. These findings led us to conclude that the SINE of hippopotamus was genetically related to a whale SINE, CHR-2 [the hippopotamus SINE was named CHR-2(hippo)] and was a retroposon derived from glutamic acid tRNA. Hipo53 and hipo95, which were the genetically most separated CHR-2(hippo) sequences in the present study, were used as a probe for dot-blot hybridization to examine the distribution of their homologous sequences among animal species. Although the distribution spectra of hipo53 and hipo95 homologous sequences in animal species differed to some extent, large amounts of both sequences were found in Hippopotamus amphibius and Globicephala macrorhynchus (whale); and small amounts in most of the animal species in Artiodactyla examined. These findings indicated that the hippopotamus and whale had more recently branched off from the clade that includes chevrotain and pecorans than the other animal species in the clade. The 30 CHR-2(hippo) sequences were aligned, and the substitution rates among the sequences were calculated with a different substitution rate model for transition and for transversion. The calculation combined with the mutation rate of the pseudogenes (r = 4.6 x 10(9)) indicated that CHR-2(hippo) sequences diversified at least 132 million years ago (Myr).

Animals↗