Biomedical subjects

C Wada

Publications and source records attributed to C Wada.

At least 55 records · Page 3Linked to original sources

PubMed1994

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.

DNA Replication↗

PubMed1994

Expression of the chemokine superfamily in rheumatoid arthritis.

The infiltration of leucocytes into the joint of rheumatoid arthritis (RA) is believed to be mediated by chemotactic factors released by activated cells. In this study, examination was made of the gene expression and production of the chemokine superfamily in RA patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation. Cultured synovial fibroblasts were found capable of expressing and producing IL-8, GRO, monocyte chemotactic and activating factor (MCAF), macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta and RANTES in response to IL-1 alpha. The expression of IL-8, GRO, MCAF, MIP-1 alpha, and MIP-1 beta was clearly shown to increase in freshly isolated synovial fluid mononuclear cells (SFMC) of RA patients, in contrast to peripheral blood mononuclear cells (PBMC) of RA patients and normal subjects. The gene expression of RANTES appeared to be the same for RA SFMC, RA PBMC, and normal PBMC. Thus, the over-expression of various chemokines may promote the recruitment of inflammatory cells into rheumatoid inflamed joints.

Arthritis, Rheumatoid↗

PubMed1994

[Detection of Candida albicans by PCR].

Detection of Candida albicans by the polymerase chain reaction (PCR) was conducted. Oligonucleotide primers to amplify a 180 bp fragment on the beta-tubulin gene of C. albicans were synthesized. PCR was conducted using standard strains (14 species and 32 strains) and strains isolated from clinical samples (8 species and 12 strains) in Kitasato University Hospital. The PCR specifically amplified DNA from C. albicans of all standard strains and neither any other Candida, other fungal, bacterial nor human DNA. In three samples of clinically isolated C. albicans strains, the positive bands were also detected. This PCR procedure is more specific and rapid than the conventional culture method and is applicable to clinical diagnosis of the C. albicans infections.

Candida albicans↗

PubMed1993

Expression of monocyte chemotactic and activating factor in rheumatoid arthritis. Regulation of its production in synovial cells by interleukin-1 and tumor necrosis factor.

OBJECTIVE: To investigate whether monocyte chemotactic and activating factor (MCAF) contributes to the accumulation of macrophages in the joints of patients with rheumatoid arthritis (RA). METHODS: MCAF was measured by radioimmunoassay. MCAF gene expression was determined by Northern blotting and reverse-transcriptase polymerase chain reaction. Recombinant human MCAF was injected into rabbit joints to evaluate the effect of MCAF on infiltration of macrophages. RESULTS: High levels of MCAF were detected in synovial fluid from patients with RA. Cells freshly isolated from synovial fluid expressed MCAF messenger RNA (mRNA). Fibroblast-like synoviocytes were found to express MCAF mRNA and to secrete MCAF in response to interleukin-1 (IL-1) and tumor necrosis factor in vitro. IL-1 also promoted MCAF gene expression in rabbit synovial tissue in vivo. MCAF caused marked infiltration of macrophages in rabbit synovial tissue. CONCLUSION: Our findings suggest that MCAF may contribute to the accumulation of macrophages in inflamed rheumatoid joints.

Arthritis, Rheumatoid↗

PubMed1993

Infrequent involvement of p53 mutations and loss of heterozygosity of 17p in the tumorigenesis of renal cell carcinoma.

Restriction fragment length polymorphism (RFLP) analysis and the polymerase chain reaction of the single-strand conformation polymorphism (PCR-SSCP) method were conducted to assess the loss of heterozygosity of chromosome 17p and mutations of the p53 gene in 30 surgical specimens of human renal cell carcinoma. Six of 29 tumors (20.6%) showed loss of heterozygosity on chromosome 17p in RFLP analysis, and in none of 21 tumors could a mutation be found on exons 5 to 8 of the p53 gene in PCR-SSCP analysis. We conclude that the p53 gene mutation does not play a role in the development of the majority of cases of renal cell carcinoma and that there may be another tumor suppressor gene on 17p.

Carcinoma, Renal Cell↗

PubMed1993

Infrequent involvement of p53 gene mutations in the tumourigenesis of Japanese prostate cancer.

A study was made of the incidence of p53 mutations in Japanese males with prostate cancer or benign prostatic hyperplasia. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) was used as a primary screening technique with gene sequencing being carried out in positive cases. Two out of 21 prostate cancers (9.5%) were found to have p53 mutations. These were stage B2 and D2 prostate cancers. No abnormalities were found in the remaining cases or benign prostatic hyperplasia. Mutations of the p53 gene would thus appear infrequent in the tumourigenesis of primary prostate cancer.

Adenocarcinoma↗

PubMed1993

Methods for detection of MDR1 mRNA expression on acute myelogenous leukemia cells.

Overexpression of the human multidrug resistance gene (MDR1) on acute myelogenous leukemia (AML) correlates with poor prognosis. We evaluated several methods for mRNA estimation to standardize simple and reliable techniques for identifying MDR1 positive leukemia among untreated AMLs in large scale studies. Northern blot detection of MDR1 mRNA suffered from low signal-to-noise ratio under the conventional conditions, that was improved mainly by removing unincorporated radioactivity. The amount of MDR1 transcripts on positive cells was estimated less than 10% of that of constitutive mRNA species. A modified method seemed useful in estimating the total amount of the MDR1 mRNA in a whole leukemic cell population, and suitable to study stock samples or for large prospective clinical trials. RT-PCR was more sensitive in detecting MDR1 mRNA than Northern blot analysis, and the very feature made it virtually impossible to exclude contamination with normal hematopoietic cells. This procedure showed that FAB M3 leukemias were essentially MDR1 negative, and there existed frequently myelodysplastic syndrome subpopulation which had excessive MDR1 transcripts. In situ hybridization of the mRNA with a FITC-labeled phosphorothioate oligonucleotide probe was visualized using flowcytometry or con-focus lightmicroscopy, enabled us to recognize the difference between multidrug resistant K562/ADM and its wild type.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Globin chain synthesis in hemolytic anemia reticulocytes. A case of hemoglobin Burke.

Peripheral red blood cells from an anemic patient were incubated with [14C]Leu and the labeled globin chains were analyzed by CM-cellulose column chromatography in 8M urea. The radioactivity almost equal to that of beta chain emerged between beta and alpha chains. The newly appeared materials were found to be derived from an abnormal hemoglobin (Hb), Hb Burke [beta 107(G9)Gly to Arg], by the sequence analyses of abnormal globin chain and beta globin mRNA from the patient.

Aged↗

PubMed1992

Binding of RepE initiator protein to mini-F DNA origin (ori2). Enhancing effects of repE mutations and DnaJ heat shock protein.

Replication of mini-F plasmid in Escherichia coli requires the plasmid-encoded RepE initiator protein and a number of host factors and is regulated by interaction of RepE with specific sequences near the replication origin, ori2. We have examined DNA binding properties of several hyperactive mutant RepE proteins with single amino acid substitutions. Plasmids carrying these (repE) mutations, unlike the parental plasmid, can replicate in bacterial hosts lacking the heat shock sigma factor (sigma 32) or deficient in the DnaK, DnaJ, or GrpE heat shock protein. Using gel-retardation assays, the mutant RepE proteins were shown to bind the ori2 repeated sequences with much increased affinities compared to the wild type RepE, whereas they bound to the repE operator with slightly reduced affinities. These results agreed well with the properties of mutant RepE proteins studied in vivo and accounted for the high RepE initiator activities and the high copy numbers of mutant plasmids. In addition, the DnaJ heat shock protein was found to markedly enhance the binding of wild type RepE to ori2 or the operator. DnaK protein with or without ATP failed to show such enhancements. Thus, among the heat shock proteins required for mini-F replication, DnaJ appears to play a major role in RepE binding to ori2 and the operator, perhaps accompanied by RepE activation.

Autoradiography↗

PubMed1992

A general transcription initiation factor, human transcription factor IID, overexpressed in human lung and breast carcinoma and rapidly induced with serum stimulation.

A general transcription factor IID which binds to the TATA box promoter element on RNA polymerase II genes regulates and initiates eukaryotic mRNA synthesis. A quantitative polymerase chain reaction procedure was developed and the human transcription factor IID (hTFIID) transcript was measured in normal human tissues, lung carcinomas, lung carcinoma cell lines, and breast carcinomas. In some normal tissues such as liver, fetal lung, and placenta, relatively low to moderate levels of hTFIID mRNA were detected. In contrast, hTFIID transcript was highly expressed in nearly all solid lung carcinomas and cell lines including both small cell lung cancer and non-small cell lung cancer. hTFIID mRNA was present to a greater extent in small cell lung cancer than non-small cell lung cancer in solid tumors and cell lines. In solid carcinomas of breast, overexpression of hTFIID was also detected. A serum induction study using a serum-starved small cell lung cancer cell line, Lu134BS, indicated hTFIID transcription to be rapidly induced at 15 min following stimulation and its response essentially similar to that of protooncogene, c-fos. These results indicate the involvement of the expression of the general transcription factor hTFIID in lung and breast carcinoma, such as being associated with poor differentiation and high mitotic activity.

Base Sequence↗

PubMed1992

Adult T-cell leukemia developing during immunosuppressive treatment in a renal transplant recipient.

We report a case of a 32-year-old male, an asymptomatic carrier of human T-cell leukemia virus type 1 (HTLV-1), who underwent a renal transplantation and developed adult T-cell leukemia (ATL) during the course of posttransplant immunosuppressive treatment. He was treated with combination chemotherapies consisting of cyclophosphamide, vincristine, doxorubicin, prednisolone, cisplatin, cytosine arabinoside, etoposide, and methyl-prednisolone, without any improvement. Bestrabucil (KM2210), a conjugate of chlorambucil and estradiol, was administered as an alternative therapy; this therapy successfully suppressed his leukemic cell growth, and partial remission was achieved. Posttransplant immunosuppressive therapy with prednisolone, mizoribine, and cyclosporin A might have been the predominant cause of the transition from an asymptomatic HTLV-1 infection to overt ATL. A careful approach is required with HTLV-1 asymptomatic carriers who need organ transplantation followed by immunosuppressive treatment.

Adult↗

PubMed1992

A continuous cell line (KK-2) from a supratentorial primitive neuroectodermal tumor.

Tumor tissue located in the occipital lobe with hemorrhage was obtained from a 19-year-old patient. Histological examination indicated it to consist of undifferentiated small, round cells without neuronal or glial differentiation, and possibly to be a type of primitive neuroectodermal tumor. The tumor cells were cultured for 3 years and a continuous cell line (KK-2) was established. KK-2 was transplantable to nude mice. With immunocytochemistry, neuron-specific enolase, protein gene product 9.5, vimentin, TUJ1 (a monoclonal antibody specific for neuron-associated class III beta-tubulin isotype) and 6H7 (a monoclonal antibody to NCAM produced by us) were detected. None of the following could be found: glial fibrillary acidic protein, S-100 protein, neurofilament and synaptophysin, calcitonin gene-related peptide, gastrin releasing peptide corticotropin-releasing factor, substance P, somatostatin, chromogranin, aromatic L-amino acid decarboxylase and tyrosine hydroxylase. The original tumor and KK-2 cells obtained after 3 years of culture and transplants in nude mice displayed essentially the same ultrastructural and immunohistochemical characteristics. KK-2 cells showed no differentiation to mature neuronal, glial or ependymal cells. This cell line may possibly serve as a useful model for studying cellular differentiation of human neuroectodermal tumors and normal neuronal development.

Adult↗

PubMed1992

In vitro microleakage at the gingival margin of porcelain and resin veneers.

This in vitro study investigated the effect of dental surface treatments on the seal of porcelain and resin laminate veneers to tooth structure. Nine groups of 20 teeth were prepared for labial veneers--six groups with the apical margin of the veneer in dentin, two groups with the veneer wholly bordered by enamel, and one group with the apical margin coincident with the apical margin of a previously placed glass ionomer restoration. The teeth were treated with a variety of dentin-bonding agents prior to restoration of eight groups with porcelain veneers and one group with Visio-Gem microfilled resin veneers. After placement, the veneers were stored in water for 4 days, then subjected to thermocycling before silver staining and sectioning. Results showed no leakage around the margins of the veneers wholly bordered by enamel. Complete leakage was found around the glass ionomer restorations, and nearly total leakage was found along the resin-dentin interface of the resin veneers. The porcelain veneers that extended onto dentin showed variable but limited leakage for four of five of the dentin-bonding treatments.

Acid Etching, Dental↗

PubMed1992

Pulmonary large cell carcinoma expressing neuroendocrine markers: the morphological, biological, and neuroendocrine features of their cell lines and surgical cases.

A cell line expressing neuroendocrine (NE) markers, designated as KTS9, was established from a human large cell carcinoma of the lung using serum-free medium, ACL-3. KTS9 cells showed morphological characteristics of large cell undifferentiated carcinoma (LCUC) and expressed some general NE markers including neuron-specific enolase (NSE), protein gene product (PGP) 9.5, neural cell adhesion molecule (N-CAM), synaptophysin and neurofilaments (NF) of 200 kd. Some cells of this cell line were positive to chromogranin-A (CG-A), but did not express Leu7 or aromatic L-amino acid decarboxylase (AADC). Such a cell line derived from LCUC with NE properties has not previously been reported. The biological and NE properties of the KTS9 cell line were compared with those of 2 surgical cases of LCUC with NE markers and of the KTA7 cell line previously reported to derive from large cell carcinoma and to possess NE markers such as alpha-hCG, PGP9.5 N-CAM and AADC. Tumor cells of 2 large cell carcinomas expressed NSE, PGP9.5, N-CAM and NF. The KTS9 and KTA7 cell lines and 2 large cell carcinomas were thus considered to be LCUCs with NE differentiation. Both lines had the morphological characteristics of LCUC, relatively short doubling time and discordant expression of NE markers, indicating them to be closely related to the variant type of small cell carcinoma cell lines and thus possibly to represent high-grade malignancy. They may be useful for examining the biological behavior and NE features of large cell-type NE tumors of the lung.

Adult↗

PubMed1992

Mini-F plasmid mutants able to replicate in Escherichia coli deficient in the DnaJ heat shock protein.

A subset of Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, is required for mini-F plasmid replication, presumably at the step of functioning of the RepE initiator protein. We have isolated and characterized mini-F plasmid mutants that acquired the ability to replicate in the Escherichia coli dnaJ259. The mutant plasmids were found to replicate in any of dnaJ, dnaK, and grpE mutant hosts tested. In each case, the majority of the mutant plasmids carried a unique amino acid alteration in a localized region of repE coding sequence and showed an increased copy number, whereas the minority contained a common single base change (C to T) in the promoter/operator region and produced an increased amount of RepE. All RepE proteins with altered residues (between 92 and 134) exhibited increased initiator activities (hyperactive), and many showed reduced repressor activities as well, indicating that this region is important for the both major functions of RepE protein. These results together with evidence reported elsewhere indicate that the subset of heat shock proteins serves to activate RepE protein prior to or during its binding to the replication origin and that the mutant RepE proteins are active even in their absence. We also found that a C-terminal lesion (repE602) reduces the initiator activity particularly of some hyperactive mutant RepE proteins but does not affect the repressor activity. This finding suggests a functional interaction between the central and C-terminal regions of RepE in carrying out the initiator function.

Bacterial Proteins↗

PubMed1992

[Detection of cytomegalovirus in urine specimens from renal transplant recipients using polymerase chain reaction].

Human cytomegalovirus (CMV) in this study, a polymerase chain reaction (PCR) assay was developed to detect human cytomegalovirus (CMV) from urine specimens taken from renal transplant patients and one congenital CMV infection. Twenty eight urine specimens of 20 renal transplant patients were analyzed by the PCR. CMV infection was detected in 11 specimens (8 recipients). The incidence (40.0%) of PCR positive in the renal transplant patients was highest among the other conventional methods including fluorescent antibody study, anti-IgM EIA and complement fixation test. In two cases, CMV infection could be identified by PCR before antibody development. The PCR procedure is more sensitive and rapid than conventional methods for the diagnosis of CMV. Rapid diagnosis of CMV infection using PCR may be useful in clinical diagnosis and have therapeutic value.

Adult↗

PubMed1991

Molecular cloning of rat cerebellin-like protein cDNA which encodes a novel membrane-associated glycoprotein.

Rat cerebellin and an apparent metabolite des-Ser1-cerebellin have been shown to be located in cerebellar Purkinje cells and the dorsal cochlear nucleus. A cDNA clone was isolated by screening a rat brain cDNA library using an oligonucleotide corresponding to rat cerebellins. The clone encodes 224 amino acid residues of a glycoprotein a part of whose sequence is virtually the same as that of cerebellins and which contains one putative transmembrane spanning domain. Neither an N-terminal cleavable signal peptide nor dibasic pair specific endopeptidase directly precedes or follows the cerebellin-like sequence. Expression of the cerebellin-like protein gene is developmentally regulated in rat cerebellum and shows tissue specific alternative splicing in the brain, adrenal gland and spleen of the rat. The results of Southern blot indicated the gene to possibly be a member of some gene family. Rat cerebellin-like protein, possibly a precursor of cerebellins, is a novel membrane-associated glycoprotein expressed in nervous, adrenal and immune systems and appears to synaptic functions in the central nervous system.

Amino Acid Sequence↗

PubMed1991

A pulmonary large cell carcinoma cell line expressing neuroendocrine cell markers and human chorionic gonadotropin alpha-subunit.

A cell line producing the neuroendocrine cell surface antigen and human chorionic gonadotropin (hCG) alpha-subunit, designated as KTA7, was established from human large cell carcinoma using a serum-free medium, ACL-3. KTA7 continued to grow in the ACL-3 medium, showing the morphological characteristics of large cell undifferentiated carcinoma. The KTA7 cells reacted with antibodies such as 6H7 and MOC1 directed against the cell surface antigens and PGP9.5 directed against a cytoplasmic protein of neuroendocrine cells but did not possess either most epithelial markers other than low-molecular-weight keratin (Cytokeratin) or neuron-specific enolase. The KTA7 cells, by immunostaining with anti-hCG subunit antibodies, were shown to produce hCG alpha- but not beta-subunit. Northern blot analysis showed KTA7 RNA to synthesize hCG alpha-subunit mRNA but not that of the hCG beta-subunit. Thus, the hCG alpha-subunit alone was independently expressed in KTA7. Chromosome analysis showed loss of alleles of chromosome 3p and 17 in KTA7 cells but not loss of 13q. KTA7 was considered to be derived from large cell undifferentiated carcinoma with neuroendocrine differentiation (large cell neuroendocrine tumor) and thus may fine use in studies on the pathobiology of large cell-type neuroendocrine tumors since it expresses at the same time marker substances of neuroendocrine differentiation and the hCG alpha-subunit.

Biomarkers, Tumor↗