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Biomedical subjects

K Gomi

Publications and source records attributed to K Gomi.

At least 163 records · Page 9Linked to original sources
PubMed1991

Cloning and molecular characterization of the acetamidase-encoding gene (amdS) from Aspergillus oryzae.

We have isolated an acetamidase-encoding gene (amdS) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus nidulans gene as a probe. The gene is located on a 3.5-kb SacI fragment and its nucleotide (nt) sequence was determined. Compared with the A. nidulans amdS gene, the coding region of A. oryzae gene consists of seven exons interrupted by six introns and encodes 545 amino acid (aa) residues. The deduced aa sequence has a high degree of homology with that of the A. nidulans acetamidase protein. Three introns (IVS-1, IVS-2, and IVS-4) exist at the same positions as those of A. nidulans amdS, whilst three additional introns (IVS-3, IVS-5, and IVS-6) are also present. There is no preference in its codon usage (G + C content in the third position of codons is 51%). Gene disruption experiments demonstrate that the resulting mutants show significantly reduced growth on acetamide-containing medium, indicating that the A. oryzae amdS gene encodes a functional acetamidase that is required for acetamide utilization. Transcriptional analysis by Northern blot reveals a 1.8-kb transcript in RNA extracted from mycelium grown in medium containing acetamide or acetate plus beta-alanine as the sole carbon and nitrogen sources.

Amidohydrolases↗
PubMed1991

Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.

The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.

Amino Acid Sequence↗
PubMed1991

Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.

The last three consecutive epidermal growth factor (EGF)-like structures of human thrombomodulin constitute the functional domain for protein C-activating cofactor activity and anticoagulant activity. Using site-directed deletion mutagenesis, we found that amino acid Asp349 of TME456, a recombinantly produced protein consisting of EGF-like structures 4, 5, and 6, is essential for retaining full protein C-activating cofactor activity. To investigate the role of Asp349 in the protein C-activating cofactor activity of human thrombomodulin, we have constructed two mutants of TMD123, a recombinantly produced protein consisting of domains D1, D2, and D3 of thrombomodulin, using site-directed point mutagenesis of the thrombomodulin coding sequence. In mutant TMD123A, the Asp349 codon was replaced with an Ala codon and in mutant TMD123E, the Asp349 codon was replaced with a Glu codon. The partially purified mutant proteins were assayed for their protein C-activating cofactor activity at various Ca2+ concentrations. TMD123 and TMD123E protein showed similar high levels of cofactor activity and similar patterns of Ca2+ dependence, while TMD123A had lower cofactor activity and did not show any Ca2+ dependence. We concluded that Asp349 in the fourth EGF-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.

Amino Acid Sequence↗
PubMed1991

Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models.

Antitumor activity of UCN-01 (7-hydroxy staurosporine), a selective inhibitor of Ca2+- and phospholipid-dependent protein kinase C, was examined in comparison with staurosporine, a nonselective inhibitor of protein kinases, on human and murine tumor cell lines which have some aberrations in cellular signal transduction. UCN-01 inhibited the growth of five tumor cell lines about 9 to 90 times less potently than staurosporine in vitro. UCN-01 showed an in vivo antitumor effect against three human tumor xenografts [epidermoid carcinoma A431 (c-erbB-1 overexpression), fibrosarcoma HT1080 (N-ras activation), and acute myeloid leukemia HL-60 (N-ras activation)], giving a minimum treated/control ratio of 0.40 (P less than 0.01), 0.17 (P less than 0.01), and 0.61 (P less than 0.05), respectively. UCN-01 also exhibited significant antitumor activity against two murine tumor models (fibrosarcoma, K-BALB and M-MSV-BALB), which activated the v-ras and v-mos oncogenes, showing a minimum treated/control ratio of 0.27 (P less than 0.01) and 0.21 (P less than 0.01). Staurosporine did not show significant antitumor activity against any of these five tumors. UCN-01 inhibited the down-modulation of epidermal growth factor receptor caused by phorbol 12-myristate 13-acetate in A431 cells at a near 50% inhibitory concentration for cell growth. These results imply that UCN-01 is a promising antitumor agent which has a novel mechanism(s) of action.

Alkaloids↗
PubMed1991

Antitumor activity of 7-N-[[2-[[2-(gamma-L-glutamylamino)ethyl]dithio]ethyl]]-mitomycin C.

Through the extensive investigation of new mitomycin C (MMC) derivatives, several compounds with disulfide at N-7 were found to show activities superior to MMC against murine Sarcoma 180 solid tumor. Among them, 7-N-[[2-[[2-(gamma-L-glutamylamino)ethyl]dithio]ethyl]]- mitomycin C (KW-2149) was selected for further evaluation of antitumor activity and toxicity in mice. KW-2149 exhibited activity superior to MMC in increasing survival of i.p. inoculated P388 leukemia-, M5076 sarcoma-, and B16 melanoma-bearing mice. KW-2149 administered i.v. also exhibited superior activity in inhibiting the growth of s.c. inoculated P388 leukemia, M5076 sarcoma, and colon 26 adenocarcinoma and in increasing survival of i.v. inoculated P388 leukemia- and M5076 sarcoma-bearing mice. Furthermore, KW-2149 remarkably increased the life span of MMC-resistant P388 leukemia- and L1210 leukemia-bearing mice. KW-2149 and MMC inhibited the growth of human tumors inoculated into nude mice. The activity of KW-2149 was prominent in human lung carcinoma Lu-65 and Lu-99, bladder carcinoma T24, and epidermoid carcinoma A431. KW-2149 was comparable to MMC in decreasing the number of WBC in the peripheral blood, and the thrombopenia induced by KW-2149 was mild and recovery was rapid. The in vitro anticellular spectrum of KW-2149 against 23 human tumor cell lines was similar to that of MMC. However, KW-2149 inhibited the growth of the cell lines at concentrations of 10- to 100-fold lower than MMC and showed efficient cytotoxicity against MMC-insensitive tumor cell lines. These included lung epidermoid carcinoma Calu-1, stomach carcinoma MKN-28, colon adenocarcinoma DLD-1, colon adenocarcinoma LoVo, bladder carcinoma HT-1197, sarcoma G-292, and melanoma SK-MEL-28 cells. These results indicate that KW-2149 bears interesting characteristics as a new anticancer drug and warrants further development.

Animals↗
PubMed1991

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae.

Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

Aspergillus oryzae↗
PubMed1991

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae.

Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T2 gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T2 revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T2 activity than wild-type strains.

Amino Acid Sequence↗
PubMed1991

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.

Alcoholic Beverages↗
PubMed1991

Sapurimycin, new antitumor antibiotic produced by Streptomyces. Producing organism, fermentation, isolation and biological properties.

In screening actinomycetes for antitumor compounds, Streptomyces sp. DO-116 was found to produce a new antitumor antibiotic sapurimycin. It is structurally related to, but distinct from, kapurimycins. The antibiotic was produced in a fermentation medium supplemented with high porous polymer resin which adsorbs antibiotic in the culture and results in an increase of titer. Active material was separated from the polymer resin by a solvent extraction procedure and isolated by repeated solvent extraction, adsorption chromatography and HPLC. Sapurimycin was active against bacteria, particularly Gram-positive organisms. It exhibited antitumor activity against leukemia P388 and sarcoma 180 in mice. Sapurimycin caused single strand breaks in supercoiled plasmid DNA in vitro. These results are discussed in comparison with data for kapurimycins.

Animals↗
PubMed1991

The glucoamylase cDNA from Aspergillus oryzae: its cloning, nucleotide sequence, and expression in Saccharomyces cerevisiae.

A cDNA for Aspergillus oryzae glucoamylase was cloned, using oligodeoxyribonucleotide probes derived from amino sequences of peptide fragments of the enzyme. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae, directed the secretion of active glucoamylase into the culture medium. The complete nucleotide sequence of the cDNA contained an open reading frame encoding 612 amino acid residues. Comparative studies with other fungal glucoamylases showed homologies of 67% with A. niger and 30% with Rhizopus oryzae of the deduced amino acid sequences. In the five conserved regions reported in other fungal glucoamylases, the levels of homologies between those regions of A. oryzae and A. niger enzymes were much higher (78-94%). A. oryzae glucoamylase contained no peptide region abundant in threonine and serine residue (TS-region), like that proposed to adsorb onto raw starch in A. awamori var. kawachii glucoamylase.

Amino Acid Sequence↗
PubMed1991

[Evaluation of [troponin-T] as a new biochemical marker for acute myocardial infarction].

Troponin (Tn) and tropomyosin (Tm) are located in the thin actin-containing filaments. Tn, a complex of three subunits (Tn-C, Tn-I and Tn-T), plays an important role in muscle contraction via calcium metabolism. In the present study, we evaluated the usefulness of a serum Tn-T assay in patients with acute myocardial infarction. The method, based on the sandwich immunoassay, was developed for the determination of serum Tn-T. The analytical precision data were good enough for routine analysis (CVs of repeatability and day-to-day reproducibility were between 3.2 to 6.2%). The values obtained on 60 healthy adults were below 0.2 ng/ml. Physical activity (mountain climbing and golf) increased serum total CK and myoglobin, but did not increase Tn-T. In myocardial infarction, serum Tn-T was up to 20 times higher than the cut off value at admission, and had a large peak at around 14 hr and another small peak at 40 to 55 hr after the onset. The ratio of Tn-T at around 14 and 32 hr (14/32) was an excellent marker for successful reperfusion. All cases of successful PTCA, had a ratio higher than 1.0.

Biomarkers↗
PubMed1991

[Clinical significance of creatine kinase isoform assay by a new reagent based on immunoinhibition method].

Clinical significance of creatine kinase (CK) isoform assay by a new diagnostic reagent based on immunoinhibition method was evaluated. In the method, a monoclonal antibody (CKM-G 01), which inhibits MM3 but not MM1 isoform, was used. Serial serum samples from 143 acute myocardial infarction (AMI) patients were examined in hospitals. Isoform ratio (I.R.: defined as follows; inhibited CK activity/uninhibited CK activity) increased most rapidly (whose peak appeared 8.5 h after onset of AMI), compared with total CK (18.3 h) and CK-MB (16.1 h). In 61 patient samples collected within 4 h from onset, I.R. in 25 samples (41%) exceeded reference interval, whereas total CK in only 17 samples (28%) did it. Reference interval of the I.R. was 0.42 +/- 0.33 (mean +/- SD) in 1,246 normal subjects. These results show that the assay of CK isoforms by the reagents based on immunoinhibition method is useful for earlier detection of AMI.

Antibodies, Monoclonal↗
PubMed1990

Antithrombotic effect of recombinant human thrombomodulin on thrombin-induced thromboembolism in mice.

Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

Animals↗
PubMed1990

The kapurimycins, new antitumor antibiotics produced by Streptomyces. Producing organism, fermentation, isolation and biological activities.

As a result of screening for antitumor agents from actinomycetes, the kepurimycins were isolated from Streptomyces sp. DO-115. The antibiotics were produced in a fermentation medium supplemented with high porous polymer resin which adsorbs antibiotics and results in an increase of titer. The active complex was isolated from the polymer resin by a solvent extraction procedure and was separated by column chromatography, into two minor and one major component, named A1, A2 and A3. The kepurimycins were active against bacteria, particularly Gram-positive organisms, and were cytotoxic to HeLa S3 human cerivical cancer cells and T24 human bladder carcinoma cells in vitro. Among the individual components of the kapurimycins, kapurimycin A3 exhibited the strongest antibacterial and cytotoxic activities. It showed a potent antitumor activity against murine leukemia P388 in vivo.

Animals↗
PubMed1990

Molecular cloning of the glucoamylase gene of Aspergillus shirousami and its expression in Aspergillus oryzae.

The glucoamylase enzyme (GAase) gene from Aspergillus shirousami was cloned and sequenced from genomic and cDNA libraries. The genomic gene was located in the 5.4 kb EcoRI fragment. The deduced amino acid sequence of GAase contained 639 amino acid residues with a relative molecular mass of approximately 68,000 daltons (non-glycosylated form). The genomic gene of A. shirousami GAase was introduced into Aspergillus oryzae. These transformants had increased GAase and raw starch degradation (RSD) activity in culture media and in rice-koji extracts. Analysis by Southern, Northern, SDS-PAGE, and Western blot techniques confirmed the foreign gene was correctly transcribed, translated, and expressed in A. oryzae.

Amino Acid Sequence↗