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Y Wada

Publications and source records attributed to Y Wada.

At least 253 records · Page 14Linked to original sources
PubMed1999

Formation of cell junctions between grafted and host cardiomyocytes at the border zone of rat myocardial infarction.

BACKGROUND: Cardiomyocyte transplantation is an innovative strategy for the treatment of heart failure after myocardial infarction. Cell junctions show diverse temporal polarization toward intercalated disks during postnatal development and exhibit altered distribution in diseased hearts. To elucidate the formation of cell junctions between grafted and host cardiomyocytes at the border zone of myocardial infarction, the 3D distribution of cell junctions was examined using immunohistochemistry and confocal microscopy. METHODS AND RESULTS: Neonatal cardiomyocytes obtained from 3-day-old rats by collagenase digestion and Percoll density centrifugation were injected into the border zones of infarction sites 10 days after coronary ligation in adult rats. At 4 to 14 days after transplantation, hearts were harvested and processed by immunohistochemistry. Antibodies against connexin43, desmoplakin, and cadherin were used to analyze the distribution of gap junctions, desmosomes, and adherens junctions, respectively. Grafted cardiomyocytes were identified by immunohistochemistry for alpha-smooth muscle actin. Grafted cardiomyocytes tended to align parallel to the host cardiomyocytes. Connexin43, desmoplakin, and cadherin were localized between grafted cardiomyocytes themselves and between grafted and host cardiomyocytes. Semiquantitative analysis revealed that all junctions showed increasing polarization to longitudinal cell termini, especially at the border of grafted and host cardiomyocytes, as time advanced from 4 to 7 days after transplantation. CONCLUSIONS: These findings indicate that grafted cardiomyocytes foster electrical pathways with host counterparts through the gap junction and suggest that the environment in infarcted hearts could influence the localization of gap junctions, desmosomes, and adherens junctions.

Animals↗
PubMed1999

Recurrent cerebral infarctions and del(10)(p14p15.1) de novo in HDR (hypoparathyroidism, sensorineural deafness, renal dysplasia) syndrome.

We report on a Japanese boy with HDR syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia) and recurrent cerebral infarctions in the basal ganglia. The patient experienced cerebral infarctions four times between age 7 months and age 20 months. Chromosome analysis of the patient demonstrated a 46,XY, del(10)(p14p15.1) de novo. This suggests that the putative gene responsible for HDR syndrome is located at 10p14-p15.1.

Adult↗
PubMed1999

Identification of natural antigenic peptides of a human gastric signet ring cell carcinoma recognized by HLA-A31-restricted cytotoxic T lymphocytes.

Peptides of human melanomas recognized by CD8+ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.

Amino Acid Sequence↗
PubMed1999

Automated determination of 5-fluorouracil and its metabolite in urine by high-performance liquid chromatography with column switching.

We report a quantitative assay of 5-fluorouracil (FU) and its metabolite, 5-fluorodihydrouracil (FDHU) in human urine by used a column-switching high-performance liquid chromatographic method. The analyses were carried out using a molecular exclusion column for sample purification, and a cation-exchange column for separation. Each sample required only 40 min to analyze, and required no preparation other than filtration. Linearity was verified up to 1000 nmol/ml (r > 0.993). The recovery of FU was 96-101%; recovery of FDHU was 96-105%. The imprecision (RSD) for FU (10-100 nmol/ml) was < 1.5%, same-day (n = 5), and < 1.8%, day-to-day (n = 5). The imprecision (RSD) for FDHU (10-100 nmol/ml) was < 3.2%, same-day (n = 5), and < 4.0%, day-to-day (n = 5). The detection limits were, respectively, 0.1 nmol/ml. We measured FU and FDHU in urine of seven cancer patients after oral administration of FU. The cumulative quantity ratio of the FDHU and FU (FDHU/FU) excreted in their urine within 120 min after FU administration was a constant value in all seven patients. Based on these results, we believe that our method provides a useful tool for evaluating FU metabolism.

Breast Neoplasms↗
PubMed1999

Establishment of a combined strategy of genetic and mass spectrometric analyses for characterizing hemoglobin mutations. An example of Hb Hoshida (beta43Glu-->Gln).

Structural analysis of mutant hemoglobins has been efficiently accomplished by a consecutive mass spectrometric strategy: molecular mass measurement of the protein to detect mutation and to determine the molecular mass change, and fragmentation analysis with collision-induced dissociation to determine the site and type of mutation. A flaw of this method is an inherent inability to detect a mutation associated with no or little change of the molecular mass. In the present study, the strategy was improved by incorporating genetic analysis prior to mass spectrometry, which confirms the resulting amino acid change and searches for possible post-translational modifications. The method was applied to an unknown mutant, and elucidated a substitution of glutamine for glutamic acid at position 43 of beta-globin subunit, the mutation of Hb Hoshida.

Amino Acid Sequence↗
PubMed1999

Casein kinase II is responsible for phosphorylation of NF-L at Ser-473.

Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo.

Amino Acid Sequence↗
PubMed1999

An inherited prion disease with a PrP P105L mutation: clinicopathologic and PrP heterogeneity.

OBJECTIVE: To clarify a clinical and neuropathologic phenotype of an inherited prion disease associated with a missense mutation at codon 105 in the prion protein (PrP) gene that was originally described as a variant of Gerstmann-Sträussler-Scheinker disease demonstrating spastic paraparesis. METHODS: Two siblings from a Japanese family are described. PrP gene analyses, neuropathologic studies with immunohistochemistry, and Western blot analysis of the PrP were performed. RESULTS: Both patients showed a missense (proline-->leucine) mutation at codon 105 and a methionine/valine polymorphism at codon 129 of the PrP gene. Clinically, Patient 1 presented with progressive spastic paraparesis, ataxia, and dementia. Patient 2, the sister of Patient 1, showed prominent action myoclonus and dementia. Neuropathologically, multiple PrP-positive amyloid plaques and diffuse PrP deposition in the deep cortical layers were found in the cerebral cortex with primarily frontal dominant atrophy in both patients. Tau-positive pathologic structures including neurofibrillary tangles, neuropil threads, and dystrophic neurites around the plaques were abundant in the brain of Patient 2. In contrast, the tau pathology was scarce in Patient 1. Western blot analysis of the brain showed different patterns of detergent-insoluble PrP fragments between the patients. CONCLUSIONS: Despite the identical codon 105 mutation and codon 129 polymorphism of the PrP gene, remarkable clinical and neuropathologic differences, and PrP heterogeneity were present between the affected siblings. The phenotypic variability might be related to PrP heterogeneity.

Amino Acid Substitution↗
PubMed1999

The gamma-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli.

The rotation of the gamma-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase (FOF1). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the gamma-subunit of the F1 sector rotated on addition of ATP. This progress enabled us to analyze the gammaM23K (the gamma-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F1 sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the gammaM23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.

Actins↗
PubMed1999

Human histamine H2 receptor gene: multiple transcription initiation and tissue-specific expression.

We have characterized the genomic structure of 12.3 kb of the 5'-flanking region of the human histamine H2 receptor gene. The multiple transcription initiation sites of the human histamine H2 receptor gene were mapped utilizing the 5'-end cap structure of mRNA. We found that a 85 bp segment (-610-(-)525 bp) immediately upstream of the initiation site exhibits a strong promoter activity in the gastric adenocarcinoma, MNK45, expressing human histamine H2 receptor. A 4.8 kb transcript of the human histamine H2 receptor gene was found in the placenta, spinal cord, lymph node and bone marrow in addition to the previously reported tissues including the heart, brain and stomach, whereas a 1.8 kb transcript was observed in almost all tissues examined. 3'-rapid amplification of cDNA ends revealed the corresponding length of the 3'-untranslated region. These results suggest that the 3'-untranslated region may be involved in the differential expression.

Base Sequence↗
PubMed1999

Pindolol potentiates the effect of fluoxetine on hippocampal seizures in rats.

Recent studies have shown that behavioral and neurochemical changes induced by selective serotonin (5-HT) reuptake inhibitors such as fluoxetine are potentiated by coadministration of a 5-HT1A receptor antagonist. The present study assessed the effects of concomitant administration of fluoxetine and a 5-HT1A receptor antagonist, pindolol, on focal hippocampal (HIP) seizures elicited by electrical stimulation in rats. A 10 mg/kg dose of fluoxetine, which was ineffective by itself, produced a significant increase in the afterdischarge threshold of HIP seizures when combined with pindolol at 10 mg/kg. The inhibitory effect of this combination was eliminated by pretreatment with parachlorophenylalanine, a depletor of brain 5-HT. These findings suggest that treatments designed to increase 5-HT neurotransmission inhibit the generation of HIP seizures. A combination of fluoxetine with a 5-HT1A receptor antagonist could thus be therapeutically useful for the treatment of depressive symptoms in patients with epilepsy.

Animals↗
PubMed1999

Sensing of cadmium and copper ions by externally exposed ADL, ASE, and ASH neurons elicits avoidance response in Caenorhabditis elegans.

We developed a quantitative assay for Caenorhabditis elegans avoidance behavior. This was then used to demonstrate that the worm moved away from toxic concentrations of Cd2+ and Cu2+, but not Ni2+, all ions that prevented development from larval to adult stages. Mutants that have structural defects in ciliated neurons (che-2 and osm-3) as well as worms with three laser-operated neurons (ADL, ASE, and ASH), showed no avoidance behavior from Cd2+ and Cu2+. These results suggest that the avoidance from Cd2+ and Cu2+ are mediated through multiple neural pathways including ADL, ASE, and ASH neurons. We hypothesize that the three sensing neurons provide increased accuracy of the sensory response and a survival advantage in the natural environment of the worm.

Animals↗
PubMed1999

Pharmacological effects of tolterodine on human isolated urinary bladder.

Tolterodine, (R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamine+ ++, is an antimuscarinic drug developed for the treatment of overactive bladder with symptoms of frequency, urgency and urge incontinence. We investigated the effects of tolterodine and its major active metabolite, DD 01 (PNU-200577), (R)-N,N-diisopropyl-3-(2-hydroxy-5-hydroxymethylphenyl)-3-phenylpropa namine, on the contractions induced by carbachol, KCl, CaCl2 and electrical field stimulation in human isolated urinary bladder smooth muscles, using the muscle bath technique. Specimens of human urinary bladder were obtained from 20 patients who underwent total cystectomy due to malignant bladder tumor. The detrusor preparations were taken from the intact part of the dome region of the bladder. Carbachol (10(-9)-10(-2) M) caused concentration-dependent contraction of human detrusor smooth muscles. Tolterodine (10(-9)-10(-6) M), DD 01 (10(-9)-10(-6) M), oxybutynin (10(-8)-10(-6) M), propiverine (10(-8)-10(-6) M), atropine (10(-9)-10(-6) M), pirenzepine (10(-8)-10(-5) M), methoctramine (10(-8)-10(-5) M) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (10(-9)-10(-6) M) caused typical shifts to the right of the concentration-response curves for carbachol, except for higher concentrations (10(-5) M) of oxybutynin and propiverine, which caused a decrease of about 30% of the maximum contractile responses to carbachol. All the slopes of the regression lines of Schild plots were close to unity, and the rank order of pA2 values was: atropine = DD 01 = tolterodine = 4-DAMP = oxybutynin > propiverine = pirenzepine > methoctramine. Tolterodine (10(-9)-10(-6) M) and DD 01 (10(-9)-10(-6) M) did not inhibit the KCl-induced (80 mM) and CaCl2-induced (5 mM) contractions, while oxybutynin (10(-8)-10(-5) M) and propiverine (10(-8)-10(-5) M) significantly inhibited the contractions. Electrical field stimulation (2-60 Hz) caused frequency-dependent contraction of human detrusor smooth muscles, which were significantly inhibited by various drugs. In the presence of 10(-6) M atropine, tolterodine and DD 01 did not inhibit the residual contractions induced by electrical field stimulation at any of the frequencies, while oxybutynin (10(-5) M) and propiverine (10(-5) M) significantly inhibited the atropine-resistant part of the contractions. The results suggest that the inhibitory effects of tolterodine and DD 01 are mediated only by their antimuscarinic action, which is equal to that of oxybutynin and significantly greater than that of propiverine, and that tolterodine and DD 01 have neither Ca2+ channel antagonist action nor inhibitory effect on the atropine-resistant part of the contractions in human detrusor smooth muscles. These findings support the usefulness of tolterodine as a therapeutic drug for overactive bladder with symptoms of frequency, urgency and urge incontinence.

Aged↗
PubMed1999

Identification and cloning of rat galectin-2: expression is predominantly in epithelial cells of the stomach.

A complementary DNA clone preferentially expressed in the gastrointestinal tract was obtained from a rat stomach library. The protein coded by the clone had a single carbohydrate recognition domain having conserved motifs for beta-galactoside binding and showed 67% amino acid identity with human galectin-2. The recombinant protein synthesized in Escherichia coli could bind to an asialofetuin column and was eluted with beta-galactopyranoside. From these observations, we named the protein rat galectin-2 coded by the cDNA. The rat galectin-2 was predominantly expressed in the epithelial cells of stomach. Thus this protein may form a mucin layer cross-linking with the beta-galactoside moiety of glycoproteins.

Amino Acid Sequence↗
PubMed1999

Detection rates of TT virus among children who visited a general hospital in Japan.

Recently, genomic DNA of the novel TT virus (TTV) was isolated from patients suffering from posttransfusion hepatitis of unknown etiology. We examined sera from 197 children who visited the Department of Pediatrics at Toyohashi National Hospital. Sera were tested for TTV DNA by seminested polymerase chain reaction (PCR) using a set of primers synthesized according to the published TTV sequence. Ten children were found to be positive for TTV (5.1%). All positive PCR products were directly sequenced in both directions using a fluorescent dye terminator cycle sequencing system. The sequences were compared by a multiple sequence alignment and a phylogenetic tree was constructed. The phylogenetic tree showed that two of the TTV isolates found in the present experiment did not belong to any of the phylogenetic groups previously reported.

Base Sequence↗
PubMed1999

Route of TT virus infection in children.

TT virus (TTV) is a novel viral agent, detected recently in non-A to E hepatitis cases. Little is known about its natural history or routes of transmission in childhood. For the detection of serum TTV DNA, semi-nested polymerase chain reaction (PCR) was carried out using TTV-specific primers and TTV nucleotide sequences were determined by the dideoxy chain-mediated termination method. Five of the 70 children studied (including 20 hepatitis B virus [HBV] carriers, 40 children born to HBV carrier mothers and 10 children born to hepatitis C virus [HCV] carrier mothers) had serum TTV DNA. Three of the 5 children had siblings (4 in total), so that a total of 9 children were studied to determine the time of initial serum TTV DNA detection. In the 8 seropositive children, the time of serum TTV DNA detection ranged from 6 to 14 months after birth, and TTV DNA persisted thereafter throughout the follow-up period. The TTV DNA-negative child was assessed most recently at 6 months of age. TTV DNA was detected in only 2 of the 4 mothers tested (families 2 and 3). When 271-bp TTV DNA fragments from each of the 8 children were sequenced, the degree of homology between siblings in families 1-3 was 100%, 99.5%, and 92.3%, respectively. The degree of homology between child-mother pairs of families 2 and 3 was 99.5-100% and 62. 6-63.9%, respectively. The distribution of different TTV strains was consistent within families, except for family 3. None of the TTV-infected children had elevated levels of alanine aminotransferase or clinical signs of liver disease.

Adolescent↗
PubMed1999

Measurement of polymorphic trinucleotide repeats in the androgen receptor gene by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Trinucleotide repeats are polymorphic in normal individuals. CAG repeats in the X-linked androgen receptor gene were counted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). A region of approximately two hundred base-pairs containing the repeats was amplified by polymerase chain reaction, then measured after a simple purification procedure. The single-charged molecular ion species was detected using 0.1 pmol of DNA sample and the number of repeats was determined from the molecular mass. The results indicated that MALDI/TOF-MS is a high-throughput alternative to polyacrylamide gel electrophoresis for precise determination of polymorphic trinucleotide repeats.

Base Sequence↗
PubMed1999

Giant solid cystic tumor of the pancreas with a fibrous septum caused by extracapsular growth in middle-aged woman: report of a case.

We report herein the case of a 57-year-old female who was admitted to our hospital for treatment of rectal carcinoma and incidentally found to have a giant cystic tumor, 20 cm in diameter, in the body and tail of the pancreas. Several imaging procedures, including ultrasonography, computed tomography, and magnetic resonance imaging, showed that this tumor was round and hypovascular, and had a septum with protuberant lesions. The capsule was 3-4 mm thick with partial calcification. A preoperative diagnosis of a solid cystic tumor (SCT) of the pancreas was made, even though these tumors are rarely found in females in their fifties. Moreover, few reports of SCT with a septum have been described. Distal pancreatectomy and splenectomy were performed, and the tumor was found to be filled with massive necrotic tissue. Microscopically, the tumor was confirmed to be a SCT with a fibrous septum. We present this case due to the atypical findings, including the age of the patient and the existence of a septum caused by extracapsular growth.

Biopsy, Needle↗