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Biomedical subjects

S Negoro

Publications and source records attributed to S Negoro.

At least 181 records · Page 10Linked to original sources
PubMed1984

Construction of hybrid genes of 6-aminohexanoic acid-oligomer hydrolase and its analogous enzyme. Estimation of the intramolecular regions important for the enzyme evolution.

Hybrids were constructed of the genes for two homologous enzymes, 6-aminohexanoic acid-oligomer hydrolase (EII, one of the nylon oligomer degradation enzymes), and its probable evolutionary antecedent (EII'). The structural genes of EII (nylB) and EII' (nylB') have 88% similarity in their nucleotide sequences, an open frame encoding a peptide of 392 amino acids, conserved restriction sites, and in vitro recombination between these genes at the corresponding restriction sites generated genes directing various hybrid enzymes. In a comparison of the EII, EII', and the hybrid enzymes, we concluded that one or more of the four amino acid alterations that occurred in the intramolecular region (between amino acids 162-257) of EII' is essential to the adaptation of the enzyme to nylon oligomer degradation, and that its effect is enhanced 20-fold by one or more further alterations in the 258-380 region. Our results also suggest that this technique is useful for improving enzyme characteristics.

Amidohydrolases↗
PubMed1984

DNA-DNA hybridization analysis of nylon oligomer-degradative plasmid pOAD2: identification of the DNA region analogous to the nylon oligomer degradation gene.

Fine structure of the gene of 6-aminohexanoic acid cyclic dimer hydrolase, one of the enzymes responsible for the degradation of the nylon oligomer (6-aminohexanoic acid cyclic dimer), on the plasmid pOAD2 harbored in Flavobacterium sp. KI72 was determined by constructing miniplasmids from plasmid pNDH5 (a hybrid plasmid consisting of pBR322 and a 9.1-kilobase-pair HindIII fragment of pOAD2 ). The 6-aminohexanoic acid cyclic dimer hydrolase produced by cells of Escherichia coli C600 harboring pNDH5 or its miniplasmid was examined immunologically and electrophoretically and was found to be identical to that of Flavobacterium sp. KI72 . A fragment of pOAD2 (17.2- to 19.1-kilobase-pair region on pOAD2 ) was detected as hybridized fragment by Southern blotting experiments, indicating the presence of the DNA region analogous to the 6-aminohexanoic acid cyclic dimer hydrolase gene on the plasmid.

Amidohydrolases↗
PubMed1984

Molecular basis of isozyme formation of beta-galactosidases in Bacillus stearothermophilus: isolation of two beta-galactosidase genes, bgaA and bgaB.

Bacillus stearothermophilus IAM11001 produced three beta-galactosidases, beta-galactosidase I, II, and III (beta-gal I, II, and III), which are detectable by polyacrylamide (nondenatured) gel electrophoresis. By connecting restriction fragments of the chromosomal DNA to plasmid vectors, followed by transformation of Escherichia coli, two beta-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were isolated. Identification of the gene products and Southern hybridization analyses with a 2.7-kilobase-pair EcoRI fragment containing the bgaA gene as probe revealed that a single bgaA gene exists on the genome and that beta-gal II and beta-gal III consist of a common subunit (the bgaA gene product; molecular weight, 120,000), but differ in their assembly (beta-gal II is a dimer, and beta-gal III is a tetramer). The bgaB gene product (molecular weight, 70,000) in Bacillus subtilis harboring pHG5 (a hybrid plasmid consisting of pUB110 and a 2.9-kilobase-pair EcoRI fragment) was estimated to be the beta-gal I protein from its heat stability. Southern hybridization and immunological testing indicated that the two genes have no homology.

Cloning, Molecular↗
PubMed1984

Carcinoembryonic antigen (CEA) levels in pleural effusions and sera of lung cancer patients.

For determining the value of carcinoembryonic antigen (CEA) levels in diagnosis of malignant tumors of the lung, the CEA levels in 187 specimens of pleural fluid and sera obtained simultaneously from patients with pleural fluid were measured. In all 70 patients with benign diseases, the CEA levels in the effusions were less than the cut-off value of 5 ng/ml (mean +/- SD: 1.44 +/- 1.01 ng/ml). In contrast, in 88 of 117 patients (75.2%) with malignant diseases, the CEA levels in the effusions were over 5 ng/ml (25.3 +/- 24.5 ng/ml) and in 58 of the 117 patients (50.4%), the CEA levels in the serum were values of 5 ng/ml or more (11.9 +/- 18.4 ng/ml). There was a significant correlation between the CEA levels in the effusions and in the sera. The CEA levels in effusions in patients with malignant lung tumors were usually much higher than those in their sera. The incidence of CEA levels of 5 ng/ml or more in both the serum and effusion was highest in the patients with adenocarcinoma. These data indicate that determination of the CEA level in effusions, when done in combination with cytological examinations, may have additional value in diagnosis of lung cancer.

Adenocarcinoma↗
PubMed1984

[Local application of anti-cancer drugs for the treatment of malignant pleural and pericardial effusion].

Pleural effusion is a common complication in patients with malignant neoplasm. A randomized controlled study of intrapleural instillation of Adriamycin (control group, 30 patients) and Adriamycin Nocardia rubra cell wall skeleton (N-CWS group, 26 patients) with tube thoracostomy was performed in 55 patients with malignant pleural effusion due to primary lung cancer. The response rates for control of pleural effusion were 73.4% in the N-CWS group and 46.1% in the N-CWS group. These results suggest that intrapleural instillation using a combination of anti-cancer agent and immunopotentiator is an effective treatment for malignant pleurisy. Cardiac tamponade secondary to cancer is a life-threatening complication requiring immediate treatment. Twenty-four patients with malignant pericardial effusion were treated by intrapericardial instillation of anti-cancer drugs, such as Carbazilquinone, Mitomycin-C or ACNU, with pericardial drainage. The range of survival time from the instillation of anti-cancer drug was 3-365 days (average days). In only 4 patients, reaccumulation of pericardial effusion was recognized. There were no serious complications with this procedure. It was considered that local instillation of anti-cancer agents with pericardial drainage was a useful therapeutic modality for malignant pericarditis.

Adenocarcinoma↗
PubMed1984

Monoclonal antibody SN2 defining a human T cell leukemia-associated cell surface glycoprotein.

We generated a monoclonal antibody, termed SN2, which defines a human T cell leukemia-associated cell surface glycoprotein, GP37, with an approximate m.w. of 37,000. This antibody was generated by using a human leukemia antigen preparation. The reactivity and specificity of SN2 were characterized by a sensitive radioimmunoassay against a variety of cultured and uncultured human cells. In selected cases, the cell specimens were tested further by indirect immunofluorescence staining. Among the various cultured malignant and nonmalignant human cell lines tested, SN2 reacted only with leukemic T cell lines, with one exception. It reacted with 10 of 11 leukemic T cell lines tested; the 10 reactive cell lines are PEER, JM, MOLT-4, CCRF-CEM, CCRF-H-SB2, RPMI 8402, DND-41, HPB-ALL, SKW-3, and HPB-MLT; the unreactive line was HUT 78. The reactive cell lines were derived from patients either with T cell-type acute lymphoblastic leukemia (the first eight cell lines), with T cell chronic lymphocytic leukemia (SKW-3), or with Japanese adult T cell leukemia-lymphoma (HPB-MLT). The unreactive cell line, HUT 78, was from a patient with Sezary syndrome. Results consistent with the above were obtained from studies in which uncultured malignant cell specimens from different cancer patients were tested against SN2; SN2 reacted only with T leukemia cells. Among various uncultured normal cell specimens tested, SN2 did not react with thymocytes, bone marrow cells, peripheral blood lymphocytes containing B and T cells, purified T cells, monocytes, granulocytes, or erythrocytes. It did, however, react with platelets.

Animals↗
PubMed1983

Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli.

The 7.7 Mdal PstI fragment of Bacillus pumilus IPO containing genes for xylan degradation, xylanase, and beta-xylosidase was inserted at the PstI site of pBR322 and cloned in E. coli C600. The hybrid plasmid thus formed was named pOXN29. The amount of xylanase and beta-xylosidase expressed in E. coli harboring pOXN29 was about 6% and 20% of the activity produced by the donor, B. pumilus. The reverse orientation of the inserted fragment resulted respectively in 5 times and 50 times increases in xylanase and beta-xylosidase productivities. Both enzymes expressed in E. coli transformants were shown to be indistinguishable from those of B. pumilus by immunological and chemical criteria. Digestion of pOXN29 with BglII produced two fragments; one was 6.7 Mdal in size and contained the whole pBR322 and the beta-xylosidase gene, and the other was 3.7 Mdal and coded for xylanase. Analysis of enzymes expressed in the transformant cells indicated that neither enzyme was secreted into the culture medium, periplasm nor membrane bound, although xylanase but not beta-xylosidase, was secreted into the medium in a B. pumilus culture.

Bacillus↗
PubMed1983

Monoclonal antibody that defines a unique human T-cell leukemia antigen.

We have generated and characterized a hybridoma monoclonal antibody, termed SN1, that defines a unique human T-cell leukemia antigen. This antibody was generated by using a human leukemia antigen preparation isolated from cell membranes of MOLT-4, a leukemia T-cell line derived from a patient with T-cell-type acute lymphoblastic leukemia (T-ALL). SN1 was characterized by a sensitive microscale radioimmunoassay using a variety of cultured and uncultured human cells. In selected cases, the cell specimens were further tested by immunoperoxidase staining and an immunofluorescence staining test. The results of the radioimmunoassay were in agreement with those of the two other tests. Among the various cultured malignant and nonmalignant cell lines, SN1 reacted only with leukemia T-cell lines derived from patients with T-ALL; it reacted with all six T-ALL cell lines tested-i.e., JM, CCRF-CEM, CCRF-H-SB2, RPMI 8402, PEER, and MOLT-4. In the case of uncultured cell specimens derived from cancer patients, SN1 reacted with four of four cases of T-ALL but did not react with specimens derived from 41 patients with other types of cancer. SN1 did not react with any normal human cell specimens tested, both cultured and uncultured. These specimens include normal lymphoblastoid cell lines, thymocytes, bone marrow cells, spleen cells, lymph node cells, peripheral blood mononuclear cells, lymphocytes containing B and T cells, purified T cells, monocytes, granulocytes, erythrocytes, and platelets. Furthermore, SN1 did not react with phytohemagglutinin-activated T cells nor with concanavalin A-activated T cells. The results show that monoclonal antibody SN1 defines a type of human leukemia antigen that is expressed on the cell surface of T-cell-type ALL cells. The results further show the usefulness of SN1 in the diagnosis of cancer patients and suggest its therapeutic potential. We designate this antigen TALLA, a T-cell ALL antigen.

Antibodies, Monoclonal↗
PubMed1983

Plasmid-determined enzymatic degradation of nylon oligomers.

The nylon oligomer (6-aminohexanoic acid cyclic dimer) degradation genes on plasmid pOAD2 of Flavobacterium sp. KI72 were cloned into Escherichia coli vector pBR322. The locus of one of the genes, the structural gene of 6-aminohexanoic acid linear oligomer hydrolase, was determined by constructing various deletion plasmids and inserting the lacUV5 promoter fragment of E. coli into the deletion plasmid. Two kinds of repeated sequences (RS-I and RS-II) were detected on pOAD2 by DNA-DNA hybridization experiments. These repeated sequences appeared five times (RS-I) or twice (RS-II) on pOAD2. One of the RS-II regions and the structural gene of the hydrolase overlapped.

Amidohydrolases↗
PubMed1983

[Conditions suitable for chemotherapy of lung cancer].

The influence of several factors for response to Chemotherapy and survival was analysed in 310 patients with inoperable lung cancer who were treated by single or combination chemotherapy. In small cell lung cancer, survival was strongly affected by the results of induction chemotherapy, and combination chemotherapy with three or four drugs was most effective. In non-small cell lung cancer, performance status and body weight loss on admission were important factors for response to chemotherapy and survival. Combinations did not improve the response rate in non-small cell lung cancer.

Adenocarcinoma↗
PubMed1982

Isopeptidase: a novel eukaryotic enzyme that cleaves isopeptide bonds.

In an attempt to clarify the regulatory mechanism that accounts for the shift of protein A24 in the mitotic cycle, we demonstrated the existence of an enzyme, provisionally termed isopeptidase, that cleaves A24 stoichiometrically into histone H2A and ubiquitin. Properties of this enzyme are (i) most eukaryotes, including mammals, amphibia, chicken, and yeast, contain isopeptidase in the cytoplasm; (ii) a significant increase in enzyme binding to chromatin occurs when cells enter mitosis; (iii) Escherichia coli does not contain isopeptidase; (iv) isopeptidase has a molecular weight of 38,000; (v) at an ionic strength that induces globular conformation of H2A, isopeptidase activity is repressed; (vi) a SH group is an essential cofactor; and (vii) most divalent cations (except Mg2+ and Ca2+) are inhibitory. In view of the stoichiometric conversion of A24 into H2A and ubiquitin by isopeptidase in vitro, A24 probably contains a Gly-Gly dipeptide in isopeptide linkage but no other intervening polypeptides. Since ubiquitin in various eukaryotes binds to protein other than H2A, and is proteolytically released, isopeptidase probably acts on isopeptide bonds in general and not uniquely on those of A24. Inasmuch as isopeptidase is present throughout the cell cycle, the level of A24 in chromatin appears to be controlled by a balance between isopeptidase and an as yet unestablished H2A-ubiquitin ligase.

Animals↗
PubMed1982

[Combination chemotherapy with peplomycin and carboquone (esquinon) for squamous cell carcinoma of the lung].

Eighteen previously untreated patients with squamous cell carcinoma of the lung were treated with a combination of a new bleomycin derivative, peplomycin and esquinon (PQ). One patient achieved a complete response (5.5%) and 5 patients a partial response (27.8%). Overall response rate was 33.3%. Median survival time of 6 patients with complete and partial response was 54 weeks and that of 12 patients with no change and progressive disease was 15 weeks. Toxicities included nausea and/or vomiting in 89%, fever in 61%, interstitial pneumonitis in 28% and leukopenia in 17%. PQ regimen appears to be effective in the treatment of squamous cell carcinoma of the lung.

Aged↗
PubMed1982

Several new monoclonal antibodies directed to human T-cell leukemia antigens.

Murine monoclonal hybridoma antibodies were generated efficiently by using a human leukemia antigen preparation which was isolated from the cell membranes of MOLT-4 (a T-leukemia cell line) by means of a novel system. The generated monoclonal hybridoma antibodies were screened and characterized by a radioimmunoassay using a variety of cell specimens as targets. The results showed that the antigen(s) defined by several of these monoclonal hybridoma antibodies is associated with human T-cell leukemia and further suggest that this antigen(s) is a new type of human leukemia-associated cell surface antigen.

Antibodies, Monoclonal↗
PubMed1981

New antigenic determinant common to human thymus leukaemia-associated antigens and common acute lymphoblastic leukaemia antigen.

In the present paper we demonstrate that there is an antigenic determinant common to two different types of human leukaemia-associated antigens, i.e. human thymus leukaemia-associated (HTL) antigens and common acute lymphoblastic leukaemia (cALL) antigen. In addition, there are distinct antigenic determinants which are unique to the HTL antigens or to the cALL antigen. The new, common antigenic determinant will be useful as a new differentiation marker of human haematopoietic cells as well as a new leukaemia-associated marker. In the present study, we used several different anti-human leukaemia antisera which include those prepared by immunizing rabbits (Nos. 7549 and 7550) with purified HTL antigens. The antisera 7549 and 7550, without any absorption, showed strong specificity as well as strong antibody activity toward leukaemia cells. These are the first anti-human leukaemia antisera, except for the monoclonal hybridoma antibodies, that show good specificity for leukaemia cells without prior absorption.

Animals↗
PubMed1981

Strong, specific anti-human leukemia antisera prepared with the use of purified cell membrane antigen.

Two rabbits immunized with 15 micrograms of a purified human thymus leukemia-associated antigen preparation and boosted once with the same amount of the antigen preparation yielded antisera that showed strong specificity for human leukemic T-cells without any prior absorptions. These antisera from the two rabbits showed a 50% killing of cells at antiserum dilutions of 5700- and 1600-fold, respectively, against JM, a leukemic T-cell line, and slightly weaker activity against MOLT-4, another leukemia T-cell line. These antisera, without any absorption, showed no or minimal reaction against two nonmalignant B-cell lines (RPMI 1788 and RPMI 8057), a leukemic non-T, non-B-cell line (NALM-16), a leukemic pre-B-cell line (NALM-1), normal peripheral blood lymphocytes, and T-cells isolated from peripheral blood lymphocytes. Antiserum 7557, which showed the higher antibody activity, was further studied by an absorption test using various human cell lines. The antiserum showed strong activity against all three leukemic T-cell lines tested, i.e., CCRF-CEM, RPMI 8402, and CCRF-HSB-2, whereas it showed no significant activity against other cell lines which included two leukemic non-T, non-B-cell lines (KM-3 and NALM-6), NALM-1 and RPMI 1788. These are the first anti-human leukemia antisera, except for monoclonal hybridoma antibodies, that showed good specificity for leukemia cells without prior absorption. The present procedure of immunizing animals with a small amount of human thymus leukemia-associated antigen preparation isolated from cell membrane will also be useful for obtaining strong, specific antisera of other cell membrane antigens.

Antibody Specificity↗