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H Anzai

Publications and source records attributed to H Anzai.

At least 55 records · Page 3Linked to original sources
PubMed1995

In situ mRNA hybridization technique for analysis of metastasis-related genes in human colon carcinoma cells.

The purpose of this study was to determine whether the expression level of several genes that regulate different steps of the metastatic process correlates with the metastatic potential of human colon carcinoma cells. The mRNA expression level for epidermal growth factor receptor (growth), basic fibroblast growth factor and interleukin-8 (angiogenesis), type IV collagenase (invasion), E-cadherin and carcinoembryonic antigen (adhesion), and the multidrug resistance gene mdr-1 (drug resistance) in the human KM12 colon carcinoma cell lines and clones with different metastatic potential was measured by Northern blot analysis and by in situ hybridization technique. Highly metastatic KM12SM and KM1214 cells growing in culture uniformly expressed high levels of epidermal growth factor receptor, basic fibroblast growth factor, and carcinoembryonic antigen mRNA, whereas cultures of low metastatic KM12C, clone 1, clone 3, and clone 6 cells displayed heterogeneous patterns of expression. KM12C (low metastatic) and KM12SM (highly metastatic) cells were implanted into the subcutis (ectopic) or the wall of the cecum (orthotopic) of nude mice. The mRNA expression level for epidermal growth factor receptor, basic fibroblast growth factor, interleukin-8, type IV collagenase, carcinoembryonic antigen, and mdr-1 was increased in the cecal wall tumors as compared with subcutaneous tumors or in vitro cultures. These data demonstrate a direct correlation between constitutive and inducible expression of several metastasis-related genes and the metastatic potential of human colon carcinoma cells.

Adenocarcinoma↗
PubMed1995

Multiparametric in situ messenger RNA hybridization analysis to detect metastasis-related genes in surgical specimens of human colon carcinomas.

We examined the expression of several genes that regulate different steps of metastasis in surgical specimens of human colon carcinomas. The expression of epidermal growth factor receptor (growth), basic fibroblast growth factor [(bFGF), angiogenesis], type IV collagenase (invasion), E-cadherin (adhesion), and multidrug-resistant (mdr)-1 (drug resistance) mRNA was examined using an in situ mRNA hybridization (ISH) technique and Northern blot analysis. Dukes' stage C and D tumors exhibited a higher level of expression (P <0.05) for bFGF, type IV collagenase, and mdr-1 mRNA than Dukes' stage B tumors. The expression level of epidermal growth factor receptor and E-cadherin did not correlate with the stage of the disease. The ISH technique revealed intertumoral heterogeneity for expression of several genes among Dukes' stage B neoplasms. In some Dukes' stage B tumors, we also found intratumoral heterogeneous staining for bFGF and type IV collagenase, with the highest expression level at their invasive edge. In Dukes' stage C and D tumors, the expression of these genes was more uniform. These results recommend the suitability of the multiparametric ISH analysis for metastasis-related genes to identify individual colon cancers with metastatic potential.

Blotting, Northern↗
PubMed1992

Synergistic cytotoxicity with 2'-deoxy-5-azacytidine and topotecan in vitro and in vivo.

Synergy, when it can be convincingly established, is an effective strategy for the development of novel drug combinations. We have evaluated the interaction between 2'-deoxy-5-azacytidine (DAC) and 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan) based on our hypothesis that DAC, through DNA hypomethylation, might increase the transcription of topoisomerase I (topo I) leading to increased sensitivity to topotecan. Five human tumor cell lines, A375 melanoma, DX-3 melanoma, DMS4C non-small cell lung carcinoma, UP-1 unknown primary adenocarcinoma, SN12C renal carcinoma, and the murine CT-26 tumor cell line, were studied. Drug interactions were assessed using the multiple drug effect analysis of Chou and Talalay (Chors, T-C, and Talalay, P. Adv. Enzyme Regul., 22:27-54, 1984.). A synergistic interaction was documented in four human cell lines and the murine CT-26 line. An antagonistic interaction was observed with the SN12C cell line. The toxicology and efficacy of this combination were analyzed using CT-26 in BALB/c mice. Various treatment schedules were studied, including: single doses of each agent; single sequential combination treatments where DAC was administered followed by topotecan 24 h later; and multiple sequential treatments where DAC and topotecan were administered on days 1, 2, 8, and 9. Efficacy studies showed that the single sequential combination of DAC (50 mg/kg) and topotecan (10 mg/kg) resulted in tumor growth delay as compared to single doses of DAC (50 mg/kg) or topotecan (10 mg/kg). When the multiple sequential combination schedule was used, the antitumor effect was more pronounced. In that experiment 50% of the control animals had tumors of 20 mm by day 28. For animals receiving a single sequential treatment with DAC and topotecan, the median time until the mean tumor size reached 20 mm was 38 days, and for the group with multiple sequential combination treatments the time was 51 days. Studies of the mechanism of the interaction showed that the activity of topotecan versus each cell line correlated with the topo I activity in nuclear extracts However, there was no correlation between topo I levels and synergy and no reproducible increase in topo I activity following exposure to DAC. Thus, while the exact mechanism of the interaction remains unclear, DAC can be effectively combined with topotecan to enhance antitumor activity.

Adenocarcinoma↗
PubMed1992

Expression of a Maize Ubiquitin Gene Promoter-bar Chimeric Gene in Transgenic Rice Plants.

We have constructed a chimeric gene consisting of the promoter, first exon, and first intron of a maize ubiquitin gene (Ubi-1) and the coding sequence of the bar gene from Streptomyces hygroscopicus. This construct was transferred into rice (Oryza sativa L.) protoplasts via electroporation, and 10 plants were regenerated from calli that had been selected for resistance to exogenously supplied bialaphos. Transgenic plants grown in a greenhouse were resistant to both bialaphos and phosphinothricine at a dosage lethal to untransformed control plants. Evidence of stable integration of the transferred gene into the genome of the regenerated primary transformant plants was obtained from Southern blot analysis. In addition, northern blot analysis indicated expression and proper splicing of the maize ubiquitin gene first intron from the primary chimeric transcript in these transgenic rice plants, and western blot analysis and enzymic assays verified expression of the active bar gene product. Apparent mendelian segregation for bialaphos resistance in T(1) progeny of primary transformants was confirmed.

Journal Article↗
PubMed1991

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus.

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

Anti-Bacterial Agents↗
PubMed1990

Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation.

An enzyme catalyzing the formation of an unusual C-P bond that is involved in the biosynthesis of the antibiotic bialaphos (BA) was isolated from the cell extract of a mutant (NP71) of Streptomyces hygroscopicus SF1293. This enzyme, carboxyphosphonoenolpyruvate (CPEP) phosphonomutase, was first identified as a protein lacking in a mutant (NP213) defective in one of the steps in the pathway to BA. The first 30 residues of the amino terminus of this protein were identical to those predicted by the nucleotide sequence of the gene that restored BA production to NP213. The substrate of the enzyme, a P-carboxylated derivative of phosphoenolpyruvate named CPEP, was also isolated from the broth filtrate of NP213 as a new biosynthetic intermediate of BA. CPEP phosphonomutase catalyzes the rearrangement of the carboxyphosphono group of CPEP to form the C-P bond of phosphinopyruvate.

Anti-Bacterial Agents↗
PubMed1990

Biochemical mechanisms of C-P bond formation of bialaphos: use of gene manipulation for the analysis of the C-P bond formation step.

One of the three C-P bond formation steps, defined as step 5 in the bialaphos (BA) biosynthetic pathway, was analyzed using a new BA non-producing mutant NP71. The mutant was derived from a BA producer by gene replacement of an unidentified region next to the gene responsible for the step 5 deficiency of the mutant NP213, obtained by conventional mutation procedures. Biochemical analysis of these two mutants indicated that NP71 was defective in the formation of carboxyphosphonoenolpyruvate (CPEP), while NP213 lacked the enzyme CPEP phosphonomutase, which catalyzed the intramolecular rearrangement of CPEP.

DNA↗
PubMed1988

The bialaphos resistance gene (bar) plays a role in both self-defense and bialaphos biosynthesis in Streptomyces hygroscopicus.

We inactivated the bialaphos (BA) resistance gene (bar) of a BA producer, Streptomyces hygroscopicus, by the gene replacement technique. The resulting BA-sensitive mutant (Bar-) was able to produce little BA but considerable amount of an intermediate demethylphosphinothricin (DMPT). The Bar- mutant was still able to convert the N-acetyl derivative (AcDMPT) of DMPT to BA. Introduction of normal bar containing plasmid restored both BA resistance and BA biosynthesis to levels as high as the parental BA producer. By contrast, introducing a multi copy glutamine synthetase gene (glnA) into the Bar- mutant restored BA resistance but not BA production. Thus, the bar gene plays a crucial role in both self-defense and a step of BA biosynthesis in the BA-producing S. hygroscopicus.

Anti-Bacterial Agents↗
PubMed1988

Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences.

We have developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. We constructed the plasmid pMSB212 which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoR I site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of known genes and to identify new genes encoded on a cloned fragment.

Chromosomes, Bacterial↗
PubMed1988

The bialaphos biosynthetic genes of Streptomyces hygroscopicus: cloning and analysis of the genes involved in the alanylation step.

We have isolated and studied the genes involved in the alanylation step in the biosynthesis of a herbicide, bialaphos which is produced by Streptomyces hygroscopicus. Three bialaphos-nonproducing mutants, NP60, NP61 and NP62, isolated from S. hygroscopicus by treatment with N-methyl-N'-nitro-N-nitrosoguanidine were defective for the alanylation step and were not restored to productivity by any locus of the gene cluster previously cloned. Three plasmids were isolated using NP60, NP61 and NP62 as recipients. The genes which restored productivity to NP61 and NP62 hybridized to the contiguous region of the bialaphos biosynthetic gene cluster. The gene cluster involved in the bialaphos production was about 35 kb long. The gene which restored productivity to NP60 did not hybridize to the bialaphos biosynthetic gene cluster. VM3 and VM4, putative alanylation blocked mutants, were derived from a bialaphos producer by gene replacement of an unidentified region of the biosynthetic gene cluster with an in vitro altered DNA sequence. The genes which restored productivity to VM3 and VM4 were located between the genes which code for phosphinomethylmalic acid synthase and demethylphosphinothricin acetyltransferase in the cluster. These results suggest that multiple genes are involved in the alanylation step.

Alanine↗
PubMed1987

Colour flow mapping of oesophagogastric varices and vessels in and around the liver with trans-oesophageal real-time two-dimensional Doppler ultrasound.

Conventional transcutaneous ultrasound examinations are often compromised by intervening intestinal or pulmonary gas and have limited resolution. Ultrasonic probes of frequencies greater than 5 MHz, which enhance resolution, cannot be used successfully on the skin surface, because they do not penetrate enough to to visualise intra-abdominal organs in most adults. To overcome these problems, we have used transoesophageal real-time two-dimensional Doppler echography. The ultrasonic probe, with a 5 MHz, curved array, was integrated into the end of a steerable insertion tube. Fifteen patients with oesophagogastric varices were examined. Oesophagogastric varices were visualised in colour in 10 patients. The direction of blood flow was determined in six patients. The flow velocity was measured quantitatively in five patients by the pulsed Doppler technique. The vessels in and around the liver were also visualised even when they could not be seen with transcutaneous ultrasonography. This technique is useful for the evaluation of both oesophagogastric varices and other abdominal vessels.

Adult↗
PubMed1987

Transcriptional regulation of bialaphos biosynthesis in Streptomyces hygroscopicus.

A DNA sequence (brpA) which regulates the expression of the genes of the bialaphos biosynthesis pathway (bap) in Streptomyces hygroscopicus was identified and characterized. A newly isolated nonproducing mutant (NP57) had a pleiotropic defect involving at least 6 of the 13 known bap genes; only the step 6 conversion could be detected. NP57 was more sensitive to bialaphos than its parent and had depressed levels of the demethylphosphinothricin acetyltransferase activity (step 10 in the pathway) which confers bialaphos resistance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of extracts of this mutant showed that it lacked proteins corresponding to steps 5 and 10. NP57 lacked mRNAs for steps 5, 10, and 13. Bialaphos productivity of NP57 was restored by transformation with a plasmid containing a 5.9-kilobase DNA fragment which was adjacent to the structural gene cluster. Subcloning experiments showed that a 1.3-kilobase fragment from this primary clone restored all the defects of NP57. We conclude that brpA can activate the transcription of the bialaphos resistance gene as well as at least six other bap structural genes.

Cloning, Molecular↗
PubMed1984

Purification and characterization of a cellulase from Dolabella auricularia.

A highly purified cellulase [EC 3.2.1.4] preparation (B beta) was obtained from a crude extract of gastric teeth of Dolabella auricularia by successive chromatographies on Sephadex G-100, DEAE-Toyopearl and CM-Toyopearl. The purified cellulase showed a single protein band on disc electrophoresis, and its isoelectric point was at pH 8.6. It contained relatively large amounts of basic amino acids and its molecular weight was estimated to be approximately 44,000 by sodium dodecyl sulfate (SDS) electrophoresis. The highest activity of this enzyme was attained at pH 6.3, but the enzyme was rather labile to heat. The activity of this enzyme was strongly inhibited by Hg2+, Mn2+, Zn2+, and Cu2+, whereas Ca2+ and Mg2+ showed no significant effect on the activity. The purified cellulase hydrolyzed sodium carboxymethyl cellulose (CMC) and phosphoric acid-swollen cellulose (swollen cellulose), as well as cellooligosaccharides and their reduction products, in an endowise fashion. It produced higher cellooligosaccharides effectively from swollen cellulose. Cellooligosaccharides with degrees of polymerization of 4-6 (G4-G6) were also hydrolyzed by the purified cellulase, but the modes of hydrolysis of these oligosaccharides were different from each other. The enzyme did not effectively attack cellooligosaccharides lower than G4. It produced G4 and G2 from G6, and G4 and glucose from G5. When these oligosaccharides were modified by reduction with sodium borotritide, the second linkage from the reducing end became, in each case, significantly susceptible to the enzyme and was preferentially cleaved.

Amino Acids↗