Biomedical subjects

K Gomi

Publications and source records attributed to K Gomi.

At least 91 records · Page 5Linked to original sources

PubMed1996

Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding alpha-glucosidase.

The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding alpha-glucosidase was determined. A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at -670 nt, -596 nt and -544 nt relative to the start codon, respectively. The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system. Deletion of the upstream half of Region III (IIIa; -544 to -529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction. Deletion of Region I and the downstream half of Region III (IIIb; -521 to -511) resulted in a significant reduction in GUS activity, but did not affect maltose induction. This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa. In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

Aspergillus oryzae↗

PubMed1996

Synthesis and antitumor activity of duocarmycin derivatives: A-ring pyrrole analogues of duocarmycin B2.

A series of the eight-substituted A-ring pyrrole derivatives of duocarmycin B2 were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. In addition, the stability of the analogues in aqueous solution was examined. The 8-H and the 8-CN compounds which cannot structurally release the cyclopropane compound (DU-86), exhibited extremely diminished anticellular activity compared with duocarmycin A (1a) or DU-86. The ethers and the sulfonates which were not converted to DU-86 under usual conditions (35 degrees C, pH 7), showed almost equal in vivo activities to that of 1a. However, their optimal doses were significantly higher than that for 1a. Most of the A-ring pyrrole analogues which can be chemically or enzymatically converted to DU-86, displayed remarkably superior in vivo antitumor activity to 1a. These results suggest that the A-ring pyrrole analogues need to chemically or enzymatically release DU-86 as an active metabolite to exhibit potent in vivo antitumor activity.

Animals↗

PubMed1996

Synthesis and antitumor activity of duocarmycin derivatives: modification of segment A of duocarmycin B2.

Several A-ring pyrrole derivatives of duocarmycin B2 were synthesized effectively from the 3-hydroxy compounds by utilizing an interesting acid-catalyzed rearrangement, their anticellular activity was preliminarily evaluated by assays of growth inhibition of HeLa S3 cells (in vitro) and antitumor activity against murine sarcoma 180 (in vivo). The 8-O-N,N-dialkylcarbamoyl derivatives of the A-ring pyrrole compound showed remarkably potent in vivo antitumor activity, superior to that of duocarmycin B2. these derivatives were subjected to further biological evaluation. They exhibited potent antitumor activity toward murine solid tumors including M5076 sarcoma, B-16 melanoma and Colon 26 adenocarcinoma. Their most noteworthy feature was their efficacy against various human xenografts including LC-6 (lung), St-4 (stomach), and Co-3 (colon).

Animals↗

PubMed1996

Molecular cloning of a genomic DNA for enolase from Aspergillus oryzae.

We have isolated an enolase gene (enoA) from Aspergillus oryzae by heterologous hybridization using the corresponding Saccharomyces cerevisiae ENO2 gene as a probe. A 2.9-kb BglII-fragment contained the entire structural gene enoA including 5'- and 3'- flanking regions. The homology between A. oryzae enoA and S. cerevisiae ENO2 genes is 66.9% when introns are removed. Genomic Southern analysis indicated that there is only one enolase gene in A. oryzae.

Amino Acid Sequence↗

PubMed1996

Immunological detection of lipopolysaccharide antigens of thermophilic campylobacters captured on polymyxin-coated polyester cloth.

Cholate-extracted lipopolysaccharide (LPS) antigens from thermophilic campylobacters were captured on polymyxin-coated polyester cloth. The captured antigens were detected by sequential reactions with rabbit anti-Campylobacter antibody, anti-rabbit IgG peroxidase conjugate and chromogenic peroxidase substrate. A polyclonal rabbit antibody elicited against a single Campylobacter-jejuni strain detected the reference strains of the twenty most frequently isolated thermophilic campylobacters in the Lior serotyping scheme. Moreover, LPS antigens of six C. Jejuni Penner serotypes fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting were recognized by four antisera prepared against homologous and heterologous Penner serotypes. The results suggest the potential application of polymyxin-cloth enzyme immunoassay for rapid detection of thermophilic campylobacters where monoclonal antibodies can be raised to possible common LPS epitopes.

Animals↗

PubMed1996

[Evaluation of image quality of medical facsimile as a radiological image recording system].

We examined the usefulness of a new medical facsimile (MFAX) system in recording and transmitting various kinds of medical images, including X-ray images and colored histopathologic images. The system consists of an image scanner, a magnetic disk for image storage, a transmission circuit and a thermal image printer. Transmission time for a FCR (Fuji computed radiography) image by super-fine mode was 6 minutes. We used ROC (receiver operating characteristics) curves to evaluate the ability of eight radiologists to detect the small simulated nodules placed on an anthropomorphic chest phantom and shown on MFAX images. The radiologists observed both the FCR films and MFAX copies and determined the presence or absence of simulated nodules using five confidence levels. The results obtained for FCR films and MFAX images showed no statistically significant difference.

Humans↗

PubMed1996

[Analysis of cyclin dependent kinase inhibitors, p16INK4a and p15INK4b gene, in acute lymphoblastic leukemias].

We examined the abnormality of p16INK4a and p15INK4b genes in 14 cases of human acute lymphoblastic leukemias (L1; 8 sample from 6 cases, L2; 10 samples from 7 cases, L3; 1 sample from 1 case) using DNA from bone marrow cells. The frequency of homozygous deletion of p16INK4a was 21.4% (3/14) and that of p15INK4b was 7.1% (1/14) and both genes were deleted in 7.1% (1/14) according to Southern blot and PCR analysis. The deletion of p16INK4a and/or p15INK4b was detected in 50% (3/6) of L1, 28.6% (2/7) of L2. The frequency of deletion was 33.3% (3/9) of B cell origin, 66.7% (2/3) of T cell origin and 0% (0/2) of nonBnonT cell origin. By PCR-SSCP analysis on exon 1 and exon 2 of p16INK4a and p15INK4b genes, we detected one case of unusual migrated band in L1 B cell origin. The base substitution, C to G, located in intron 1 of p15INK4b, 9 base upstream of intron 1-exon 2 boundary, was determined by DNA sequencing analysis. Deletion of p16INK4a and/or p15INK4b gene may contribute to etiology of ALL.

Adolescent↗

PubMed1996

[Multi-drug resistance of serovar E strains in clinically isolated Pseudomonas aeruginosa and analysis of genome pattern].

We analyzed the in vitro antibiotic susceptible pattern and serovar for 132 strains of pseudomonas aeruginosa (98 from inpatients and 34 from outpatients) isolated at Showa University hospital from September to December of 1993. The ratio of resistant strains was 38.6% for carbenicillin, 12.1% for piperacillin, 12.9% for ceftazidime, 12.1% for cefsulodin, 17.4% for cefoperazone, 9.1% for cefclidin, 24.2% for cefpirome, 7.6% for imipenem, 17.4% for amikacin, 16.7% for aztreonam, 89.4% for minocycline and 33.3% for ofloxacin. The incidence of resistance against minocycline, carbenicillin, ofloxacin was markedly high. Among the 132 tested strains of Pseudomonas aeruginosa, 16 were multi-drug resistant strains (i.e., resistant to more than 8 drugs out of 12 drugs), and 14 of the multi-drug resistant strains (87.5%) were isolated from inpatients. Multi-drug resistant strains were isolated most frequently from urine specimens. The antibiotics that remained effective against the multi-drug resistant strains included amikacin, imipenem, cefclidin and cefsulodin. The serovar of 116 strains (resistant to up to 7 drugs) was 27 for G, 24 for B, and 19 for E, and 15 of the 16 multi-drug resistant strains showed serovar E. Restriction enzyme SpeI digestion analysis (using pulse field electrophoresis) of P. aeruginosa having serovar E revealed common 650kb and 540kb bands in multi-drug resistant strains. The genome pattern was quoted for serovar E strain according to the restriction enzyme digestion pattern and was shown to be useful for tracing the route of infection.

Drug Resistance, Microbial↗

PubMed1996

[Performance study of teleradiology network systems with CRT monitors: ROC analysis of an observation study of simulated lung nodules].

PURPOSE: To evaluate the diagnostic performance of commercially available CRT monitors used in a teleradiology system by determining the rate of detection of simulated lung nodules. MATERIALS AND METHODS: Three types of CRT monitors were tested in the observation study. They had matrix sizes of 1024 x 768(16 inches, color), 1024 x 768(20 inches, color) and 1600 x 1125(24 inches, black and white). Twenty chest radiographs were obtained by Fuji computed radiography(FCR) of an anthropomorphic chest phantom with ten simulated nodules on its surface. These FCR films were digitized by a film digitizer with 125 DPI(1024 x 1024 matrix sizes)and 12 bit gray scales, and the image data were transferred from Shinshu University Hospital to other hospitals where interpretation was carried out by the radiologists. Ten radiologists of three hospitals were asked to interpret independently both the original FCR films and the images shown on the CRT monitors and to indicate the presence or absence of simulated nodules on the images by using a five-category rating scale. Receiver operating characteristics(ROC)curves were generated, and the results of interpretation on the FCR films and CRT monitors were compared. RESULTS: Performance of the all readers was slightly better with the CRT monitors than on the FCR films, although the differences were not statistically significant. There were no statistically significant differences in performance depending on the type of CRT monitor. CONCLUSION: Performance of the CRT monitors was comparable to that of FCR radiography in terms of interpreting the simulated lung nodules.

Computer Communication Networks↗

PubMed1995

Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors.

We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes.

Base Sequence↗

PubMed1995

Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus.

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)- geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.

Amino Acid Sequence↗

PubMed1995

Synthesis and antitumor activity of various 6-demethylmitomycins and 6-demethyl-6-halomitomycins.

A series of 6-demethylmitomycins and 6-demethyl-6-halomitomycins having various mitomycin skeletons were synthesized, taking into account the electronic effect toward the quinone moiety and the partition coefficients. Treatment of enones 15 and 16 with selenenamide or N-halosuccinimide-Et2NH afforded the 6-demethyl intermediates 17, 18, and 21-24 via the tandem Michael addition/retro-Mannich reaction sequence. Subsequent conversions into the mitomycin skeletons resulted in the formation of the desired derivatives 7a-c, 8a-c, 11a-c, and 12a,b. These mitomycin derivatives including 3a-c and 4a-c were evaluated for their anticellular activity against HeLa S3 cells and antitumor activity against Sarcoma 180 in mice. The anticellular activity of 1 and 3a-c depends on the substituent at the C-6 position and the order of increasing activity is H < CH3 < Br < Cl. A similar tendency was observed in their antitumor potency (ED50). The activities of 9 and 11a-c also follow a pattern similar to that of 1 and 3a-c. Compounds 4b,c, 8b,c, and 12b having both a halogen at the C-6 position and a methoxy group at the C-7 position did not show the activities because of the instability of the compounds. Interestingly, a correlation between the anticellular activity (IC50) and the partition coefficients (log kappa') determined by HPLC was observed within the compounds studied except the unstable compounds, while their antitumor activity (ED50 or T/C) did not correlate with the quinone reduction potential (E1/2). These results would indicate the importance of the C-6 substituents and the mitomycin skeletons for exhibiting both anticellular and antitumor activities.

Animals↗

PubMed1995

Characteristics of the antitumor activity of M-16 and M-18, major metabolites of a new mitomycin C derivative KW-2149, in mice.

The cell growth inhibitory activity, antitumor activity and toxicity of M-16 and M-18, the major metabolites of a new mitomycin C (MMC) derivative KW-2149, in mouse and human were compared with those of KW-2149 or MMC in vitro and in vivo. The growth inhibitory activity of M-18, a symmetrical disulfide dimer, against human uterine cervix carcinoma HeLa S3 cells was almost equivalent to that of KW-2149 and their IC50 values were about 10-fold smaller than that of MMC. The activity of M-16, a methyl sulfide form, was almost equivalent to that of MMC. The cell-killing activity of MMC and M-16 was augmented in the hypoxic condition, whereas that of KW-2149 and M-18 was reduced. M-16 also exhibited almost equivalent activities to MMC in vivo in terms of many biological profiles, i.e., antitumor activity against murine P388 leukemia, ascitic or solid B16 melanoma or human lung carcinoma xenograft L-27, and bone marrow toxicity in mice. These in vitro and in vivo results indicate that the antitumor activity and toxicity of KW-2149 might not be mediated by M-16 in mice. On the other hand, M-18 exhibited almost equivalent activities to KW-2149 in these regards, suggesting the involvement of M-18 in the biological activities of KW-2149. However, the small values of the area under the curve of M-18 in mice make this unlikely. Thus the biological activities of KW-2149 in mice are not explained by the M-16 or M-18 concentration in plasma and are postulated to be manifested by KW-2149 itself.

Animals↗

PubMed1995

In vitro effect of chromium and other trace metals on mouse hepatotoxicity induced by carbon tetrachloride exposure.

Using primary cultured mouse hepatocytes, in vitro study was performed to discuss the effect of Cr(III) and several other trace metals, Cr(VI), Mn(II), Zn(II), Co(II), Cu(II), Ni(II), and Ga(III) on acute liver damage induced by CCl4 exposure. 1) The LDH activity 60 min after CCl4 exposure increased dose-dependently with CCl4 concentrations in all of the trace metal pretreatment groups, except for the Cr(VI) pretreatment group, which showed a significant protective effect even after 30 min of CCl4 exposure. 2) LDH leakage was not observed 10 min after CCl4 exposure at 3 or 5 mM, while lipid peroxidation was increased dose-dependently with CCl4 concentrations in all groups except the Cr(VI) pretreatment group, in which the production of peroxidated lipid was significantly inhibited. 3) Similarly to the pretreatment with Cr(VI), LDH leakage 30 min after exposure to 5 mM CCl4 was inhibited by pretreatment with such antioxidants as N,N'-diphenyl-p-phenylenediamine or DL-alpha-tocopherol. 4) The Cr(VI) uptake was about 50% of the added amount, whereas the Cr(III) uptake was only 5% of the added amount. 5) 90% or more of the intracellular chromium was reduced to Cr(III) 10 min after Cr(VI) treatment. The results suggested that the in vitro protective effect of pretreatment with Cr(VI) was due to a rapid reduction of Cr(VI) to Cr(III), and the radical scavenger-like effect of the produced Cr(III) was the same effect as in vivo Cr(III); it therefore suggests that Cr(III) contributes to protective effect on CCl4-induced hepatotoxicity.

Animals↗

PubMed1995

Synthesis and antitumor activity of duocarmycin derivatives.

A series of duocarmycin B2 derivatives, modified at the phenolic hydroxyl group to ester, carbonate and carbamate, was synthesized. Antitumor activity of these analogs was preliminarily evaluated by assays of growth inhibition of HeLa S3 cells (in vitro) and antitumor activity against murine sarcoma 180 (in vivo). The stability of the compounds under aqueous conditions was examined, and we found a correlation between antitumor activity in vivo and stability in aqueous solution, that is, the more stable derivatives exhibited higher antitumor activity. Among these derivatives, the N,N-dialkylcarbamoyl analogs exhibited both improved antitumor activity and higher stability compared with duocarmycin B2. These analogs were subjected to further biological evaluation and they expressed broad-spectrum activity toward murine solid tumors M5076, Colon 26 and Colon 38, and human xenografted carcinoma MX-1.

Animals↗

PubMed1995

Cloning and nucleotide sequence of the calmodulin-encoding gene (cmdA) from Aspergillus oryzae.

A cDNA and genomic gene encoding calmodulin were isolated from Aspergillus oryzae using a part of the calmodulin gene from A. nidulans as a hybridization probe. The gene was in a 3.4-kb SphI fragment and Southern-blot analysis of genomic DNA suggested the existence of a single copy of the calmodulin gene in A. oryzae. The nucleotide sequence analysis showed that the gene consists of five introns and six exons. Although the nucleotide sequence homology with that of A. nidulans was not so high (68%), the deduced amino acid sequence was 100% and 84% identical with calmodulin of A. nidulans and chicken, respectively. The cDNA encoding A. oryzae calmodulin was expressed under the control of the GAL1 promoter in the calmodulin null mutant (cmd1) of yeast, Saccharomyces cerevisiae, and could function as a calmodulin gene.

Amino Acid Sequence↗

PubMed1995

Nucleotide sequence and expression of alpha-glucosidase-encoding gene (agdA) from Aspergillus oryzae.

We have isolated an alpha-glucosidase(AGL)-encoding gene (agdA) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus niger gene as a probe. Southern hybridization analysis showed that the agdA gene is on a 5.0-kb ScaI fragment and there is a single copy in the A. oryzae chromosome. Comparison with the A. niger agdA gene indicated that the agdA gene contains three putative introns from 52 to 59 nucleotides long, and that it encodes 985 amino acid residues. The deduced amino acid sequence of A. oryzae AGL is 78% homologous with the A. niger AGL. The high degree of homology with the amino acid sequence bordering the putative catalytic residue of a number of AGL enzymes, and this enzyme suggests that Asp492 is a catalytic residue of A. oryzae AGL. The cloned gene was functional. Transformants of A. oryzae containing multiple copies of the cloned agdA gene showed a 6-16 fold increase in AGL activity. Like the Taka-amylase A and glucoamylase genes of A. oryzae, expression of the agdA gene was induced when maltose was provided as a carbon source, but expression was not induced by glucose. This result suggested that cis-element(s) involved in maltose induction may be also present in the agdA promoter region.

Amino Acid Sequence↗

PubMed1995

Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae.

A genomic DNA encoding ribonuclease (RNase) T1 from Aspergillus oryzae was cloned using a synthetic oligonucleotide probe. The cloned gene (designated rntA) encoded functional RNase T1, since an A. oryzae transformant with multiple copies of the rntA gene showed higher RNase T1 activity (over 200 times) than a transformant with a vector. A cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) with primers corresponding to the 5' terminus and 3' terminus of the reading frame of the rntA gene. Nucleotide sequencing analysis of both DNAs found that RNase T1 had a prepro-sequence consisting of 26 amino acids and the rntA gene had only one intron (114 bp) in the region encoding the signal sequence. The A. oryzae transformant with cDNA controlled by the amyB promoter also showed higher activity (over 300 times), indicating that the cloned cDNA encoded functional RNase T1. On the other hand, the Saccharomyces cerevisiae transformant with cDNA controlled by the GAL1 promoter could not grow on a medium containing galactose. These results suggests that A. oryzae may have a protection mechanism from RNase T1.

Amino Acid Sequence↗