Biomedical subjects

C Wada

Publications and source records attributed to C Wada.

At least 37 records · Page 2Linked to original sources

PubMed1998

A catalytic antibody that accelerates the hydrolysis of carbonate esters. Prediction of the binding-site structure of the substrate.

Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of VL and VH regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [14C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS-PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed.

Amino Acid Sequence↗

PubMed1998

[Muscle atrophy in isolated ACTH deficiency].

We analyzed muscle area in CT and muscle pathology in a patient with isolated ACTH deficiency who started with the difficulty of elevation of both arms. Cortisol treatment resulted in full recovery from severe muscle atrophy and contracture of major joints. Change of volume of major muscles in arm, thigh and calf was followed. Major muscles were identified in CT and the area of each muscle was calculated with computer assistance. The increase of total muscle area in sequential 3 times in CT was up to 74% after prednisolone treatment. This indicates that the deficiency of cortisol resulted in 42% reduction of muscle volume. This also suggests that reduction of muscle volume induces the limitation of range of motion of shoulder joint. ATPase of muscle biopsy revealed the influence on fiber type proportion; type 1 : type 2A : type 2B = 29.6 : 6.0 : 64.4% and 35.7 : 17.6 : 46.7% in pre-treatment and post-treatment of cortisol, respectively. Mean diameters of muscle fibers in type 1, type 2A and type 2B was 41.8, 41.8, 39.1 microns and 46.2, 44.0, 37.2 microns in pre-treatment and post-treatment of cortisol, respectively. These suggest that deficiency of glucocorticoid introduces the reduction of the activity of the motor neurons innervating type 1 and type 2A muscle fibers.

Adrenocorticotropic Hormone↗

PubMed1997

The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control.

The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription. Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function. Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208). The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-à-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers. Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers. On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization. Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication.

Bacterial Proteins↗

PubMed1996

The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli.

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50 degrees bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaA complex, much as has been documented for oriC dependent replication.

Bacterial Proteins↗

PubMed1996

Microsatellite instability in transitional cell carcinoma of the urinary tract and its relationship to clinicopathological variables and smoking.

To determine whether microsatellite instability is involved in the development of transitional cell carcinoma (TCC) of the urinary tract, a microsatellite instability assay was carried out using PCR with 9 microsatellite loci. Thirty-eight TCC samples (30 patients with bladder cancer, 5 with renal pelvic tumors and 3 with ureteral tumors) and 1 lymph node with metastasis were examined. Microsatellite instability was found in 8 of 38 tumors examined, and 3 showed alterations in more than 2 microsatellite loci. All 8 tumors were beyond grade 2 and stage pT2 advanced tumors. Stages pT1-2 and pT3-4 patients differed significantly. Microsatellite instability was greater in smokers than non-smokers, but the differences were not significant. Microsatellite instability in TCC of the urinary tract is rare in superficial tumors but more common in invasive tumors. Microsatellite alterations would thus appear to occur, and possibly be importantly involved, in the tumorigenesis of urinary tract TCC.

Carcinoma, Transitional Cell↗

PubMed1996

[Nucleotide sequences at intron 6 and exon 7 junction of fibroblast growth factor receptor 2 and rapid mutational analysis in Apert syndrome].

Apert syndrome, acrocephalosyndactyly Type I, is an autosomal dominant craniosynostosis comprising acrocephaly, facial dysmorphism and severe syndactyly of the hands and feet. Missense mutations at codons 252 and 253 at 5'-end on exon 7 of fibroblast growth factor receptor (FGFR) 2 have been identified in a large number of patients with Apert syndrome. In this study, nucleotide sequences on the intron 6 were determined by vector ligation-PCR and direct sequencing. Five DNA samples from sporadic Apert syndrome were examined by non-RI SSCP and direct sequencing using a primer pair of intron 6 and exon 7. All cases of the syndrome showed abnormal banding pattern in the SSCP and missense mutations from Ser to Trp at codon 252 of the FGFR2 gene. The non-RI SSCP and direct sequencing of the FGFR2 exon 7 from genomic DNAs may be a useful and rapid molecular means for clinical diagnosis of Apert syndrome.

Acrocephalosyndactylia↗

PubMed1996

[Frequent missense mutations of fibroblast growth factor receptor (FGFR) gene families in craniofacial syndromes in Japanese patients].

Craniofacial syndromes, including Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome, Apert syndrome and achondroplasia, have been indicated that syndromes were associated with mutations of fibroblast growth factor receptor (FGFR) gene families. In this report, seven Japanese patients with craniofacial syndromes, three Crouzon syndromes and four achondroplasias, were analyzed on FGFR2 and FGFR3 genes by non RI-SSCP (single strand conformation polymorphisms) and direct sequencing. Missense mutations of the FGFR3 exon 10, at codon 380 in two sporadic cases and codon 375 in two familial cases, were detected in all cases of achondroplasia. Mutations of the FGFR2 were noted in Crouzon and Apert syndromes. One of three Crouzon syndromes has a missense mutation at codon 342 on exon 9. Highly frequent mutations were clustered within some localized regions of the FGFR genes in craniofacial syndromes. Alterations in these receptors due to missense mutations would thus appear closely involved in pathogenesis of craniofacial syndrome. The non RI-SSCP and direct sequencing of the FGFR genes, shown in this report, may be an appropriate approach for diagnosis of these syndromes with extensive clinical application.

Base Sequence↗

PubMed1996

[Analysis of mutations and heteroplasmy at mitochondrial DNA 11778 using non-RI single strand conformation polymorphisms in Leber's hereditary optic neuropathy].

Leber's hereditary optic neuropathy(LHON) is a maternally inherited mitochondrial disease of an acute or subacute bilateral loss of central vision. G to A substitutions at nucleotide position 11778 in mitochondrial DNA(mt DNA) have been identified in approximately 40% to 90% of patients. In this study, regions containing mt DNA 11778 mutations were analyzed by polymerase chain reaction(PCR), non-RI single strand conformation polymorphisms(SSCP) and direct sequencing. In 26 visually affected patients, mt DNA 11778 mutations were detected in 9 patients (36.4%). In one pedigree of a LHON patient(L-6), four unaffected family members had heteroplasmy of the 11778 mutation using non-RI SSCP. Ratios of the heteroplasmy between wild type and mutant mt DNAs can be detected in non-RI SSCP and accurately quantified by video densitometric analyzer. Two types of novel polymorphisms, 11696 G to A and 11719 A to G, in the mt DNA region were also found in this non-RI SSCP analysis. Non-RI SSCP is an efficient and accurate method for diagnosis of mt DNA 11778 mutations and quantifying heteroplasmy in patients with LHON and pedigrees.

Base Sequence↗

PubMed1995

Microsatellite instability in prostate cancer.

Assessment of the genetic instability of a microsatellite has indicated a new mechanism in human carcinogenesis. Examination was made to determine whether microsatellite instability is associated with the onset of prostate cancer. Twenty-nine DNA samples from 24 primary prostate cancer, two metastatic lymph-node and three benign prostatic hypertrophy patients were used. Differences in unrelated microsatellites for tumor and normal DNA were detected in nine of 24 (37.5%) cases. Seven of 11 (63.6%) with poorly differentiated adenocarcinomas and seven of 15 (46.7%) stage D metastatic patients showed somatic instability in a number of microsatellites. Statistically significant differences in well to moderately differentiated tumors and poorly differentiated cancer (P = 0.015, Chi-square test), were detected but not for stages A-C and D (P = 0.2311). Genetic alterations would thus appear to be rare in low grade and/or early prostate cancers but more common in high grade and/or advanced prostate cancers.

DNA Replication↗

PubMed1995

Successful resection of a large hepatoblastoma in a young adult: report of a case.

Hepatoblastoma (HB) rarely occurs in adults, and very few cases of successful resection have been documented. We report herein the unusual case of a 22-year-old, otherwise healthy woman with no history of liver disease who presented with upper abdominal pain and hepatomegaly. Tests for hepatitis B virus (HBV) and hepatitis C virus (HCV) were negative, but the AFP was mildly elevated at 77 ng/ml, the normal being < 20. There was no evidence of liver cirrhosis on either the laboratory or histologic examinations. A well-demarcated solid mass of 14 cm in diameter, which was lobulated and partly necrotic, was detected in the liver by computed tomography (CT). The lesion was echogenic on ultrasound, slightly hypodense on CT, and mildly hypervascular on arteriogram. The entire tumor was resected by extensive hepatectomy preserving only the lateral segment and part of the posterior segment of the liver. Histologically, the neoplasm was diagnosed as a pure epithelial HB of the fetal type. Following the operation, the patient has been well and free of recurrence for 38 months, maintaining low alpha-fetoprotein (AFP) levels at around 5 ng/ml. To our knowledge, this is the longest reported survival of an adult following surgical resection of an epithelial HB.

Adult↗

PubMed1995

In-vivo induction of monocyte chemotactic and activating factor in patients with chronic renal failure.

BACKGROUND: Monocyte chemotactic and activating factor (MCAF) is a novel inflammatory cytokine belonging to the chemokine superfamily and stimulates chemotaxis and activation of monocytes. Increased production of inflammatory cytokines has been shown in patients with end-stage renal disease (ESRD). This study was thus conducted to determine plasma MCAF in patients with ESRD. METHODS: Plasma levels of MCAF were determined by ELISA. Gene expression of MCAF in PBMC was assessed by RT-PCR followed by southern blot hybridization. RESULTS: Plasma MCAF in 72 patients with long-term haemodialysis (HD) (162.4 +/- 58.2 pg/ml) and eight uraemic patients not yet dialysed (167.6 +/- 57.7 pg/ml) was found to exceed significantly the level in 24 normal subjects (86.0 +/- 19.4 pg/ml). MCAF before HD session in long-term HD patients was the same whether HD was carried out with either cellulosic (CUP) or synthetic (PMMA) membrane dialysers. Intradialytic increase in plasma MCAF during a single HD session was observed in both patient groups dialysed with CUP or PMMA membranes. The results of RT-PCR analysis indicated that haemodialysis stimulates the gene expression of MCAF in PBMC in vivo. CONCLUSIONS: The present results indicate that increased levels of plasma MCAF may promote the activation of monocytes in patients with ESRD.

Adult↗

PubMed1995

DNA-binding domain of the RepE initiator protein of mini-F plasmid: involvement of the carboxyl-terminal region.

The RepE initiator protein (251 residues) is essential for mini-F replication in Escherichia coli and exhibits two major functions: initiation of DNA replication from ori2 and autogenous repression of repE transcription. Whereas the initiation is mediated by RepE monomers that bind to the ori2 iterons (direct repeats), the autogenous repression is mediated by dimers that bind to the repE operator, which contains an inverted repeat sequence related to the iterons. We now report that the binding of RepE to these DNA sites is primarily determined by the C-terminal region of this protein. The mutant RepE proteins lacking either the N-terminal 33 (or more) residues or the C-terminal 7 (or more) residues were first shown to be defective in binding to both the ori2 and the operator DNAs. However, direct screening and analysis of mutant RepEs which are specifically affected in binding to the ori2 iterons revealed that the mutations (mostly amino acid substitutions) occur exclusively in the C-terminal region (residues 168 to 242). These mutant proteins exhibited reduced binding to ori2 and no detectable binding to the operator. Thus, whereas truncation of either end of RepE can destroy the DNA-binding activities, the C-terminal region appears to represent a primary DNA-binding domain of RepE for both ori2 and the operator. Analogous DNA-binding domains seem to be conserved among the initiator proteins of certain related plasmids.

Amino Acid Sequence↗

PubMed1995

Infrequent involvement of mutations on neurofibromatosis type 1, H-ras, K-ras and N-ras in urothelial tumors.

The neurofibromatosis type 1 (NF1) gene is considered a tumor-suppressor gene whose product acts upstream of ras. The ras gene is an oncogene very commonly detected in human cancers and consists of three families, H-ras, K-ras and N-ras. These genes are converted to active oncogenes by point mutations in codon 12, 13, or 61. Examination was made of the mutations of these genes in 39 urothelial malignant tumors (31 bladder cancer, 6 renal pelvic tumor, and 2 ureter tumors) using polymerase chain reaction single-stranded conformation polymorphism and direct sequencing methods. Three of 39 (7.7%) cases showed mobility shifts in the ras family gene but no point mutations in NF1 and N-ras genes could be detected. Mutations were found in 1 case in H-ras at codon 13 (GGT-GTT/GGT) and K-ras at codon 12 in 2 cases (GGT-GCT/GGT, GGT-GTT/GGT). All 3 cases had progressed far beyond grade 2 and stage pT2. It follows from the above that NF1 and ras gene mutations are infrequent in the pathogenesis of urothelial tumors.

Base Sequence↗

PubMed1995

[Molecular diagnosis of a kindred with novel mutation of methylmalonyl-CoA mutase gene using non-RI SSCP].

A deficiency of methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, which is inherited as an autosomal recessive disease and is characterized by accumulation of precursors and abnormal derivatives of methylmalonyl-CoA in body fluids. Abnormal splicing with 13 base pairs (bp) insertion at MCM exons 2 and 3 junction in MCM transcripts and a homozygous point mutation, g to a transition, on 5 bp downstream exon 2 were detected in a proband with methylmalonic acidemia. The parents in the kindred were heterozygous carriers of the g to a transition in MCM intron 2. Non-RI single strand conformation polymorphisms (SSCP) was conducted to devise for analysis of this MCM mutation. This non-RI SSCP is considered to be useful diagnostic means with high potential for extended clinical application.

Adult↗

PubMed1995

[Molecular differential diagnosis of herpes virus using common primer pairs: detection of HSV-1, HSV-2, VZV and CMV by the PCR].

Differential diagnosis of herpes virus species is often required in various clinical courses, such as transplantation and immunocompromised patients. Pairs of common primers which enabled sufficient amplifying and differential diagnosis of herpes virus species were designed and nested PCR using the common primer pairs was conducted to evaluate for its clinical application. The PCR was shown to provide amplification and differential diagnosis of HSV-1, HSV-2, VZV and CMV. The results of the PCR were consistent with those from fluorescent antibody of herpes virus culture and isolation. From clinical vesicular specimens, the PCR enabled the direct detection of HSV-1, HSV-2, and VZV. The PCR using the common primer pairs offers speed in the differential diagnosing of herpes virus.

Base Sequence↗

PubMed1995

p53 mutations and prognosis in bladder tumors.

The incidence of loss of heterozygosity on chromosome 17p and p53 gene mutations was assessed in 43 bladder tumor patients. Histological findings, cigarette smoking and prognosis were examined for possible correlation with the presence or absence of loss of heterozygosity on 17p and p53 mutations. Of 20 informative cases 10 (50.0%) showed loss of heterozygosity of 17p13, including 9 (90.0%) with disease beyond stage pT2. The p53 mutations were detected in 20 of 43 patients (46.5%), including 9 (95.0%) with disease beyond grade 2 and 17 (85.0%) with cancer beyond stage pT2. The incidence of p53 gene mutations was not significantly influenced by habitual smoking but G:C to T:A substitutions, often observed in lung cancers, were detected only in mutations from smokers (5 of 10 or 50%, p < 0.05). Groups with and without loss of heterozygosity showed essentially the same results, while significant differences were found for groups with grades 1 and 2 to 3 (p < 0.05) cancer, stages pT1 and pT2 to 4 (p < 0.01) disease, and with and without p53 gene mutations (p < 0.01, Cox-Mantel test). Genetic alternation in chromosome 17p and p53 mutations would, thus, appear to occur more frequently in high grade and invasive bladder tumors. Cigarette smoking may possibly be a determining factor of mutations of the p53 gene in bladder tumors. Our results indicate that an unfavorable prognostic factor may possibly be linked not only to histopathological findings but the presence of a p53 mutation in bladder tumors as well. Accordingly, mutations of the p53 gene may be deeply involved in late events of tumorigenesis and possibly useful as ideal molecular markers for prognosis in bladder tumors.

Adult↗

PubMed1994

Genomic instability of microsatellite repeats and mutations of H-, K-, and N-ras, and p53 genes in renal cell carcinoma.

Thirty-six primary renal cell carcinoma samples and one metastatic lymph node DNA sample were examined for mutations of H-, K-, and N-ras and p53 genes, and genomic instability at (AC)n, (CA)n.(GT)n, and (TA)n.(GT)n repeats. No mutations were noted for H-, K-, and N-ras genes and only 2 of all the samples (5.6%) showed mutations at exon 8 of the p53 gene. Differences in unrelated microsatellites for tumor and normal DNA were detected in 9 (25.0%) of the cases examined. Somatic alterations in seven microsatellites, D3S1228, D3S643, D5S107, LPL5GT, D9S63, D17S261, and DCC, were found in 1 (2.8%), 3 (8.3%), 2 (5.7%), 5 (14.7%), 3 (8.3%), 3 (8.3%), and 3 (8.3%) cases, respectively. Five of 26 (19.2%) clear cell type and 4 of 10 (40.0%) non-clear cell type patients showed DNA instability. Two of 11 (18.2%) grade 1, 5 of 20 (25.0%) grade 2, and 2 of 5 (40.0%) grade 3 patients showed abnormal patterns. One of 2 (50.0%) stage pT1, 4 of 24 (16.7%) stage pT2, and 4 of 10 (40.0%) stage pT3 patients were shown to have microsatellite instability. In 4 of 9 alteration-positive cases (44.4%), mutations in multiple microsatellites were observed. Alterations in microsatellite instability may be more common in non-clear cell type, high-grade, and high-stage renal cell carcinoma patients.

Base Sequence↗

PubMed1994

Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia.

Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor-suppressor genes. Investigation of genomic instability of microsatellites has indicated a new mechanism for human carcinogenesis in hereditary nonpolyposis colorectal cancer and sporadic cancer and this instability has been shown to be related to inherited predisposition to cancer. This study was conducted to determine whether such microsatellite instability is associated with the evolution of chronic myelogenous leukemia (CML) to the blast crisis. Nineteen CML patients clinically progressing from the chronic phase to accelerated phase or blast crisis and 20 other patients in the CML chronic phase were studied. By polymerase chain reaction assay, DNAs for genomic instability in five separate microsatellites in chromosome arms 5q (Mfd27), 17p (Mfd41), 18q (DCC), 3p (CI3-9), and 8p (LPL) were examined. Differences in unrelated microsatellites of chronic and blastic phase DNAs in 14 of 19 patients (73.7%) were demonstrated. Somatic instability in five microsatellites, Mfd27, Mfd41, DCC, CI3-9, and LPL, was detected in 2 of 19 (10.5%), 8 of 19 (42.1%), 11 of 19 (57.9%), 4 of 17 (23.5%), and 4 of 17 (23.5%) cases. In 10 of 19 cases (52.6%), genetic instability in at least two of five microsatellites was observed and was categorized as replication error (RER+) phenotype. CML evolution cases with myeloid, lymphoid, and mixed phenotypes and the blast crisis and accelerated phase showed somatic instability in a number of microsatellites. No alterations in leukemic cells at the chronic phase could be detected in any microsatellites. These data indicate instability of microsatellites (RER+) but not familial predisposition to possibly be a late genetic event in the evolution of CML to blast crisis. In the microsatellite of the DCC gene, complicated alterations in band patterns caused by instability as well as loss of heterozygosity (LOH) were observed in 13 of 19 cases (68.4%): instability in 9 cases, instability plus LOH in 2 cases, and only LOH in 2 cases. These highly frequent alterations in microsatellites, including instability and LOH, suggesting that secondary events due possibly to loss of fidelity in replication and repair machinery may be significantly associated with CML evolution.

Adult↗