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Biomedical subjects

F Ishikawa

Publications and source records attributed to F Ishikawa.

At least 127 records · Page 7Linked to original sources
PubMed1987

Active H-ras and N-ras in rat fibrosarcomas induced by 1,6-dinitropyrene.

The activated oncogenes in rat fibrosarcomas induced by subcutaneous injection of 1,6-dinitropyrene (1,6-DNP), an environmental mutagen-carcinogen, were examined by NIH3T3 cell transfection assay and Southern blot analysis. DNAs from two of eleven fibrosarcomas, 1,6-DNP6 and 1,6-DNP9T, induced transformants which contained rat specific repetitive sequences. Three primary transformants, obtained with DNA from 1,6-DNP6, and two secondary transformants of one of these primary transformants tested, contained rat H-ras. DNA from 1,6-DNP9T; a fourth transplant of 1,6-DNP9, also induced transformants. All the primary transformants with DNA from 1,6-DNP9T, and three secondary transformants of one of them, contained rat N-ras. The DNA from the original tumor, 1,6-DNP9, however, did not give any transformants by several transfection assays. Activation of N-ras oncogene during serial transplantations is discussed.

Animals↗
PubMed1987

Activated N-ras oncogene in a transformant derived from a rat small intestinal adenocarcinoma induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole.

DNAs from five intestinal adenocarcinomas induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole, which is present in broiled fish, were subjected to transfection assay using NIH3T3 cells as recipients. The DNA from only one adenocarcinoma induced a morphologically transformed focus. Rat N-ras sequences were detected in the primary transformant and in three tested secondary transformants. In the activated N-ras oncogene, a G----T transversion at the first letter of codon 12 was detected. The original tumor DNA did not hybridize with the oligonucleotide representing the mutated allele, but did hybridize with the one representing the normal allele. From these data we concluded either that the activation of the N-ras oncogene had occurred during the transfection or that the activated N-ras oncogene had been present in a minor population of cells in the original tumor.

Adenocarcinoma↗
PubMed1987

Rat c-raf oncogene activation by a rearrangement that produces a fused protein.

In a previous study, activated rat c-raf was detected by an NIH 3T3 cell transfection assay, and a rearrangement was demonstrated in the 5' half of the sequence of the gene. In the present study, the cDNAs of normal and activated rat c-raf were analyzed. Results showed that the activated c-raf gene is transcribed to produce a fused mRNA, in which the 5' half of the sequence is replaced by an unknown rat sequence. This mRNA codes a fused c-raf protein. The normal and activated c-raf cDNAs were each connected to the long terminal repeat of Rous sarcoma virus and transfected into NIH 3T3 cells. Only the activated form had transforming activity. We conclude that the rearrangement is responsible for the activation of c-raf.

Amino Acid Sequence↗
PubMed1987

The complete primary structure of the rat A-raf cDNA coding region: conservation of the putative regulatory regions present in rat c-raf.

A-raf gene was first detected by low-stringent hybridization with v-raf and was reported as a possible oncogene. We have cloned rat A-raf cDNA and determined the complete nucleotide sequence over its long open reading frame. Alignment of rat A-raf- and c-raf-deduced amino acid sequences shows 59.4% homology. Both the ATP binding site and the kinase domain are conserved in A-raf as in c-raf. Furthermore, three regions highly conserved between A-raf and c-raf were identified in the 5'-half region of c-raf which has been shown to be deleted in the activated form of c-raf. These regions were speculated to be the candidates of the regulatory domain controlling the protein kinase activity of raf products. Southern blot analysis shows that genes homologous to A-raf and c-raf are present as independent loci in Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae and Candida boidinii. A-raf was expressed in several tissues and cell line at various amounts.

Amino Acid Sequence↗
PubMed1986

Activation of rat c-raf during transfection of hepatocellular carcinoma DNA.

Rat c-raf was found to be activated in a transformant obtained with DNA of a hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline. This activated c-raf was cloned in a cosmid vector and actively transforming clones were obtained. Comparison of the restriction maps of this activated c-raf and cloned normal rat c-raf revealed a recombination in the 5'-terminal region of the activated form of this gene. The recombined DNA was shown to be actively transcribed and possibly to form a fused mRNA with c-raf, which is slightly smaller than normal c-raf mRNA. Since this recombination was not detected in the original tumor by Southern blot analysis, it presumably occurred during transfection.

Animals↗
PubMed1986

Effects of various antibiotics on antibody-producing cells of mouse spleen.

The inhibitory effect of various beta-lactams on antibody production in the spleen was evaluated in mice immunized with sheep red blood cells by Cunningham's method. With a few exceptions, cephem derivatives were more effective than the penicillins tested in inhibiting the production of antibodies in the spleen of mice. This tendency was especially marked with cefsulodin, cefmenoxime, cefotiam and ceftazidime. However, no effect was obtained with 6-aminopenicillanic acid or 7-aminocephalosporanic acid. Our findings suggest that the difference among various beta-lactams in inhibitory effect on antibody production resulted from the difference in activities of beta-lactams with different side chains at the 6- or 7-positions.

Animals↗
PubMed1986

Cysteine-rich regions conserved in amino-terminal halves of raf gene family products and protein kinase C.

The predicted amino acid sequences of rat c-raf and A-raf products were subjected to homology search against other known amino acid sequences. We found that characteristic, cysteine-rich regions present in the amino-terminal halves of c-raf and A-raf products had striking homologies with the cysteine-rich regions present in protein kinase C. These cysteine-rich regions had been shown to be deleted in the activated raf products and in the catalytically active protein kinase C produced by proteolysis. These facts suggest the significance of the cysteine-rich regions of raf products as well as those of protein kinase C in the regulation of kinase activities.

Amino Acid Sequence↗
PubMed1986

Activation of rat and human c-raf(-1) by rearrangement.

Activated c-raf(-1) gene was found in three transformants obtained by transfecting DNAs from rat hepatocellular carcinoma, metastasis of human colon cancer in mesocolon and normal mucosa from a different colon cancer patient. Rat and human activated c-raf(-1) genes were cloned into cosmid vectors; restriction enzyme mapping revealed both activated c-raf(-1) genes to have rearrangement in the center of the normal form of the gene, and the upstream sequences were replaced by unrelated sequences. Using genomic DNA fragments located immediately downstream of the recombination points, the activations of all these c-raf(-1) were shown to have occurred during the transfection process. The recombination points in both the rat and human clones isolated were located in the intron between exons 7 and 8, and nucleotide sequencing around these recombination points showed there to be an inverted repeat which could be involved in inducing in vitro recombination. Nucleotide sequencing of rat and human c-raf(-1) cDNAs revealed the upstream sequences, recombined to the 3' half of c-raf(-1), to be expressed as fusion mRNAs; the production of fused proteins was predicted from a long open reading frame, which is in-frame with the kinase domain encoded from the 3' half of the c-raf(-1) gene. There is a cysteine clustering region in an N-terminal region of the c-raf(-1) product deduced from the nucleotide sequence, and this cysteine clustering region was found to be highly homologous to that present in an N-terminal region of protein kinase C, although, in the latter cysteine clusters are present in duplicate. From analogy with the activation mechanism of protein kinase C, the N-terminal region of serine/threonine kinase coded by the c-raf(-1) gene is suggested to be a regulatory part of the enzyme activity, and it proposed that the replacement or truncation of this regulatory part could be the mechanism whereby c-raf(-1) is activated.

Amino Acid Sequence↗
PubMed1985

A new solid support for sandwich enzyme immunoassays of human immunoglobulin G.

Two antibodies were prepared for use in a sandwich enzyme immunoassay of human IgG. Completely purified guinea pig anti-human IgG was labelled with beta-D-galactosidase (EC 3.2.1.23), using a heterobifunctional cross-linker named GMBS. Partially purified anti-human IgG was immobilized on a new solid support: Amino-Dylark balls. Optimal conditions for immobilizing the antibody, using glutaraldehyde as the coupling reagent, were studied in detail. With the enzyme-labelled antibody and the solid-phase anti-human IgG, a sandwich enzyme immunoassay of human IgG with a lower limit of detection at 10.5 pM (0.3 ng/tube) was developed. A comparative study of the EIA method and a laser nephelometric method showed a good correlation. The specificity of the assay was excellent: all 4 types of IgG tested showed the maximum 0.0001%; human IgA, IgM and albumin possessed the maximum 0.54% in their cross-reactivity values with human IgG.

Animals↗
PubMed1985

Activated c-raf gene in a rat hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline.

A rat hepatocellular carcinoma, IQ7, induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) gave two transformants of NIH 3T3 cells on DNA mediated gene transfer. One of these transformants was examined further and secondary and tertiary transformants were obtained. The secondary transformant was tumorigenic in nude mice. The activated oncogene in this primary transformant was identified as rat c-raf by Southern blot analysis.

Animals↗
PubMed1985

Activation of K-ras and oncogenes other than ras family in rat fibrosarcomas induced by 1,8-dinitropyrene.

Oncogenes of fibrosarcomas of rats, induced by subcutaneous injection of 1,8-dinitropyrene (1,8-DNP), were examined by NIH 3T3 cell transfection assay and Southern blot analysis. Transformants containing rat specific repetitive sequences were obtained with DNAs of 4 fibrosarcomas, 1,8-DNP1, 1,8-DNP2, 1,8-DNP3 and 1,8-DNP7. A transformant, 1,8-DNP2-2, induced by DNA of a fibrosarcoma, 1,8-DNP2, and 7 secondary transformants derived from it contained rat K-ras sequences. Another transformant, 1,8-DNP2-1, induced by the same sarcoma did not have a ras family oncogene. This indicates that the sarcoma, 1,8-DNP2, has at least 2 transforming genes. The transforming genes of 6 other transformants derived from 3 sarcomas did not contain ras family or neu transforming genes.

Animals↗
PubMed1985

Cyclic guanidines. 17. Novel (N-substituted amino)imidazo[2,1-b]quinazolin-2 ones: water-soluble platelet aggregation inhibitors.

A series of novel 1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one derivatives substituted with a secondary amino group has been prepared and tested for the activities of inhibiting platelet aggregation in rats in vitro and ex vivo. Most of the compounds were found to be the potent inhibitors of platelet aggregation. Some of the active compounds were soluble in water and effective via iv infusion in rats. Structure-activity relationships have indicated that a lipophilic secondary amino group located at position 6 or 7 contributed to retention of potent activity. Among the compounds studied, 7-piperidino-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2- one (13 g,DN-9693) was the most favorable compound.

Animals↗