Biomedical subjects

T Miki

Publications and source records attributed to T Miki.

At least 541 records · Page 30Linked to original sources

PubMed1994

A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways.

Transfection of NIH3T3 cells with an osteosarcoma expression cDNA library led to the appearance of foci of morphologically transformed cells which were found to harbor a novel oncogene, ost. The ost product was activated by truncation of the N-terminal domain of the ost proto-oncogene and was highly tumorigenic in nude mouse assays. The proto-ost cDNA, isolated subsequently, encodes a predicted protein of 100 kDa containing DH (Db1 homology) and PH (pleckstrin homology) domains. Ost is mainly phosphorylated on serine and localized in the cytoplasm. Purified Ost protein catalyzed guanine nucleotide exchange on RhoA and Cdc42 among the Rho and Ras family members tested, indicating that Ost can activate these small GTP-binding proteins. Ost did not detectably associate with RhoA or Cdc42, but interacted specifically with the GTP-bound form of Rac1, suggesting that Ost can function as an effector of Rac1. These results suggest that Ost is a critical regulatory component which links pathways that signal through Rac1, RhoA and Cdc42. Of the tissues examined, expression of ost was the highest in brain and could be localized to neurons and alpha-tanycytes, suggesting that Ost may participate in axonal transport in these specialized cells.

3T3 Cells↗

PubMed1994

Recognition DNA sequence of a novel putative transcription factor, BCL6.

The BCL6 gene involved in the 3q27 translocation associated with B-cell lymphomas encodes a novel Cys2-His2 zinc finger protein. We generated a fusion protein of glutathione S-transferase and zinc finger domain of BCL6 to determine recognition sequences of BCL6 with polymerase chain reaction using random oligonucleotides of 26 bases as a ligand. A consensus of 14 nucleotides consisting of (T/A)NCTTTCNAGG(A/G)AT was identified in the recognition sequences. In a gel mobility shift assay, the probe containing the 14-nucleotide recognition sequence formed a complex with the fusion protein and nuclear proteins from Burkitt's cell lines overexpressing the BCL6 transcripts. The consensus sequence was protected from the digestion by nuclease in a DNase I footprinting assay. In conclusion, BCL6 may be involved in tumorigenesis by binding to the consensus sequences of the other genes.

Base Sequence↗

PubMed1994

Regional difference in the neurotoxicity of ochratoxin A on the developing cerebral cortex in mice.

Pregnant mice were treated intraperitoneally with 3 mg/kg of ochratoxin A on day 10 of gestation. They were allowed to give birth and the offspring were killed at 6 weeks of age for observation. Prenatal exposure to ochratoxin A caused microcephaly in offspring. Their body weight, brain weight, cortical thickness and numerical densities of neurons and synapses in somatosensory and visual cortex were examined. The mice exposed in utero to ochratoxin A showed a significant deficit in brain weight compared to the age-matched control, but there was no significant difference in body weight between these two groups. The cortical thickness showed a significant decrease in both somatosensory and visual cortex. Normal control mice had about 66,000 neurons/mm3, while age-matched ochratoxin A-treated mice had about 91,000 neurons/mm3 in somatosensory cortex. There was a significant increase in OA-treated group. However, there was no significant difference in the numerical density of neurons in visual cortex. On the other hand, there was no significant difference in the numerical density of synapses in both somatosensory and visual cortex. The somatosensory cortex of control mice had about 13,000 synapses per neuron, whereas ochratoxin A-treated mice had about 9,400 synapses per neuron. In the visual cortex, no significant difference was seen in synapse-to-neuron ratios. The discrepancy in the numerical density of neurons and synapse-to-neuron ratios between the somatosensory and visual cortex might derive from a time difference in cortical neurogenesis.

Aging↗

PubMed1994

Molecular cloning and expression of the acidic fibroblast growth factor receptors in a rat parathyroid cell line (PT-r). Parathyroid cell-specific calcium-dependent change of ligand accessibility and covalent attachment of heparan sulfate glycosaminoglycan to the receptors.

We have previously identified two fibroblast growth factor (FGF) receptors with higher affinity for acidic FGF rather than basic FGF in a rat parathyroid cell line (PT-r). Carbohydrate analyses of the receptors suggested the presence of three different types of FGF receptors, a 150-kDa glycoprotein receptor, a approximately 150-kDa heparan sulfate-proteoglycan receptor, and a 130-kDa glycoprotein receptor (Sakaguchi, K., Yanagishita, M., Takeuchi, Y., and Aurbach, G.D. (1991) J. Biol. Chem. 266, 7270-7278). Here, we have cloned two isoforms of the FGF receptors from PT-r cells; one with two immunoglobulin (Ig)-like domains (clone a), and the other with an additional Ig-like domain and an acidic box (clone b). They showed highest homology to the mouse and human keratinocyte growth factor receptors among the FGF receptors reported. Clones a and b had one and three possible glycosaminoglycan attachment sites, respectively. Heparitinase treatment of PT-r cells transfected with clone a suggested that the protein for the 130-kDa glycoprotein receptor was encoded by clone a, and that the same protein also served as a core protein for the approximately 150-kDa heparan sulfate-proteoglycan receptor. Heparan sulfate glycosaminoglycan attachment to the 150-kDa receptor encoded by clone b was not detectable by the same enzyme treatment. Site-directed mutagenesis (from Ser to Ala) studies of the consensus sequence for the attachment of glycosaminoglycans further supported the presence of covalently attached heparan sulfate glycosaminoglycan in the approximately 150-kDa heparan sulfate-proteoglycan receptor. These receptors overexpressed in PT-r cells changed ligand accessibility or apparently translocated after changing extracellular calcium concentrations in a manner similar to the native receptors in PT-r cells (Sakaguchi, K. (1992) J. Biol. Chem. 267, 24554-24562), whereas those expressed in CHO-K1 or NIH/3T3 cells did not. These findings strongly suggest that the two FGF receptor isoforms cloned here represent the acidic FGF receptors that we reported earlier. A subpopulation of the receptors carries heparan sulfate glycosaminoglycan covalently attached to the core protein, and the change in ligand accessibility in response to the shift in ambient calcium concentration is specific to the parathyroid cells.

Amino Acid Sequence↗

PubMed1994

Identification of DNA topoisomerases involved in immediate and transient DNA relaxation induced by heat shock in Escherichia coli.

The linking number of plasmid DNA in exponentially growing Escherichia coli increases immediately and transiently after heat shock. The purpose of this study was to search for DNA topoisomerases that catalyze this relaxation of DNA. Neither introduction of a topA deletion mutation nor treatment of cells with DNA gyrase inhibitors affected the DNA relaxation induced by heat shock. Thus, DNA topoisomerase I and DNA gyrase are apparently not involved in the process. However, the reaction was inhibited by nalidixic acid or by oxolinic acid in the topA mutant and the reaction was resistant to nalidixic acid in a topA mutant carrying, in addition, the nalA26 mutation. These results are interpreted as indicating that both DNA topoisomerase I and DNA gyrase are involved in the DNA relaxation induced by heat shock.

DNA Topoisomerases, Type I↗

PubMed1994

Temperature shift-up leads to simultaneous and continuous plasmid DNA relaxation and induction of DnaK and GroEL proteins in anaerobically growing Escherichia coli cells.

We previously reported that plasmid DNA in Escherichia coli cells growing under aerobic conditions relaxed immediately and transiently after heat shock (Mizushima, T., Natori, S. and Sekimizu, K., Mol. Gen. Genet. (1993) 238, 1-5). We next examined DNA relaxation and induction of heat shock proteins after heat shock in cells growing under anaerobic conditions. DNA in these cells relaxed rapidly (2 min) after heat shock (42 degrees C), as was the case with aerobically growing cells, but full superhelicity was not recovered. The relaxed state of DNA topology was maintained for 60 min after heat shock. Induction of DnaK and GroEL proteins, which was transient in aerobically growing cells, was continuous in anaerobically growing cells. Therefore, induction of heat shock proteins correlated with DNA relaxation in both aerobic and anaerobic conditions.

Aerobiosis↗

PubMed1994

[Recurrent dislocation of the elbow].

A case of a recurrent dislocation of the elbow joint in a 13-year-old female is described. The patient sustained a medial epicondylar fracture of the humerus and consulted us four months later. There was no deformity but marked lateral instability of the elbow was recognized. It could be fully flexed and hyperextended ca 20 degrees. The elbow joint could be easily dislocated by pronation of the hyperextended elbow. Roentgenogram demonstrated an apparent non-union of the medial epicondylar fracture. The fragment was repositioned so that the medial collateral ligament became taut. There was no recurrence of the dislocation ten months postoperatively.

Adolescent↗

PubMed1994

A human oncogene of the RAS superfamily unmasked by expression cDNA cloning.

As an approach to identify human oncogenes, we generated an expression cDNA library from an ovarian carcinoma line. A potent transforming gene was detected by transfection analysis and identified as TC21, a recently cloned member of the RAS gene superfamily. A single point mutation substituting glutamine for leucine at position 72 was shown to be responsible for activation of transforming properties. While the cDNA clone possessed high transforming activity, the ovarian tumor genomic DNA, which contained the mutated TC21 allele, failed to induce transformed foci. Thus, expression cDNA cloning made it possible to identify and isolate a human oncogene that has evaded detection by conventional approaches.

Amino Acid Sequence↗

PubMed1994

Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.

Adult↗

PubMed1994

Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma.

Amino Acid Sequence↗

PubMed1994

Expression complementary DNA library transfer establishes mrp as a multidrug resistance gene.

The emergence of drug-resistant cancer cells is a major obstacle to cancer treatment. Resistant cells often display a multidrug-resistant phenotype that reduces the promise of combination chemotherapy, the classic approach to the prevention of drug resistance. mdr1, a member of the ABC cassette superfamily of transporters which encodes an energy-dependent drug efflux pump, is the only gene known to confer the multidrug-resistant phenotype. Other multidrug resistance mechanisms must exist, since cell lines which have this phenotype in the absence of mdr1 overexpression have been described. We report here the application of a novel approach involving expression complementary DNA library transfer to the identification of drug-resistant genes. Using this approach we establish that mrp, a member of the ABC cassette superfamily of transporters, is capable of conferring a multidrug-resistant phenotype. This approach should be useful in the identification of other novel resistance genes.

3T3 Cells↗

PubMed1994

Identification and sequence of an accessory factor required for activation of the human interferon gamma receptor.

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-gamma receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-gamma.

Amino Acid Sequence↗

PubMed1994

Formation of alpha-helix 88-98 is essential in the establishment of higher-order structure from reduced lysozyme.

Lys96 and Lys97 in lysozyme are located at the C terminus of alpha-helix 88-98. The positive charges of these residues are supposed to stabilize the helical structure, and these residues are conserved as the basic amino acids among c-type lysozymes. The renaturation rate of reduced mutant lysozyme, where both Lys96 and Lys97 were mutated together to Ala, was slower than that of native lysozyme at pH 8.0 and 37 degrees C by SH-SS interchange reactions. In order to investigate the reason, the peptide fragment 36-105 (where we can obtain information of the interaction between helix 88-98 and Trp62 and Trp63 residues) was prepared. CD spectra were compared between peptide fragment 36-105 and its acetylated form, where the positive charges of Lys96 and Lys97 were eliminated, and it was elucidated that the displacement of positive charges at the C terminus of the helix caused the shift of the advantageous structure of the fragment from alpha-helix to coil. Moreover, we obtained evidence that there was interaction of the helix with Trp62 and/or Trp63, which maintained a thermodynamically stable higher-order structure. Therefore, these results suggest that the formation of alpha-helical structure in 88-98 is a significant factor in the establishment of native structure from reduced lysozyme.

Amino Acid Sequence↗

PubMed1994

Membrane potential-linked reversed electron transfer in the beef heart cytochrome bc1 complex reconstituted into potassium-loaded phospholipid vesicles.

The cytochrome bc1 complex purified from beef heart mitochondria was incorporated into potassium (K+)-loaded phospholipid vesicles by a cholate dialysis method to study the reverse reaction of electron transfer in the complex. The reduction of cytochrome b in the presence of sodium ascorbate was observed on addition of valinomycin to the K(+)-loaded proteoliposomes in a medium containing no external KCl; it was followed by the gradual oxidation. Nigericin accelerated the reoxidation of reduced cytochrome b, indicating that a K+ diffusion potential (negative inside) induced the reduction of cytochrome b. The extent of the cytochrome b reduction depended on the magnitude of the diffusion potential across the liposomal membranes, and its maximal reduction was attained at more than 210 mV of the diffusion potential. It was cytochrome b562 that was reduced during the establishment of the K+ diffusion potential in the presence of ascorbate, and about 90% of cytochrome b562 was estimated to be reduced. Antimycin A and myxothiazol inhibited the diffusion potential-induced reduction of cytochrome b562, and ubiquinone was proved to be essential for the reversed electron transfer. The K+ diffusion potential also induced the partial reduction of cytochrome b566 when cytochrome b562 had previously been reduced with ascorbate plus tetramethyl-p-phenylenediamine. These results were interpreted well based on the Q cycle scheme which assumed the energy-dependent reduction of ubiquinone at center o. Dicyclohexylcarbodiimide, which did not perturb the ability of proteoliposomes to generate the K+ diffusion potential, inhibited the energy-dependent reduction of cytochrome b562 without a significant loss in the catalytic activity of the complex. The half-inhibition was brought about by 200 mol of dicyclohexylcarbodiimide/mol of cytochrome c1. These results strongly suggest the coupling of a proton flow with the reversed electron transfer in the bc1 complex.

Animals↗

PubMed1994

Molecular cloning of the breakpoint for 3q27 translocation in B-cell lymphomas and leukemias.

Reciprocal exchanges between chromosomal region 3q27 and three loci of the Ig genes have been reported in cases of B-cell type non-Hodgkin's lymphoma. We have cloned a region containing a breakpoint junction of 3q27 from a cell line established from a patient with Burkitt's lymphoma carrying t(3;22)(q27;q11). The region cloned was shown to contain an Ig lambda light chain gene fused to a gene on chromosome 3q27. This finding was subsequently confirmed by fluorescence in situ hybridization. Extra nucleotides were present at the joining site. The heptamer-like and nonamer-like sequences separated by an intervening 24 bp were present in the region corresponding to the breakpoint of 3q27, suggesting that a misrecombination in Ig gene rearrangement may be involved in the translocation. Southern blot analysis with a 3q27-specific probe showed rearrangements in three additional patients with B-cell malignancies with the t(3;14)(q27;q32). The breakpoints of all four cases clustered within a limited 3-kb region on chromosome 3q27. The region of 3q27 involved in the translocation was designated as the BCL5 locus. The transcripts from the BCL5 locus were detected in normal tissues and hematopoietic cell lines, and the increased expression of transcript of aberrant size was detected in the established cell line carrying t(3;22). These observations suggest that a gene located at 3q27 is involved in the translocation and that its deregulation plays a role in the malignant transformation of B cells.

Base Sequence↗

PubMed1994

Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Krüppel-like zinc-finger protein.

Chromosomal translocations involving band 3q27 are the recently described nonrandom cytogenetic abnormalities in B-cell malignancies. We have previously cloned the breakpoint region of 3q27, designated as the BCL5 locus, from the B-cell line carrying the t(3;22). The cDNA for the BCL5 gene was cloned from the human liver cDNA library. The nucleotide sequencing analysis showed that the BCL5 gene encodes a potential transcription factor containing six repeats of the Cys2-His2 zinc-finger motif resembling the Drosophila segmentation gene Krüppel. The calculated molecular weight was 78.8 kD, which was supported by an in vitro transcription and translation experiment. A part of the sequence was essentially identical to that of a genomic fragment, ZNF51, previously reported to be located at 3qter. The translocation occurred in the 5' region of the BCL5 gene, and the protein-coding exons were fused to the Ig-lambda gene in a head-to-head configuration in the cell line carrying t(3;22). The BCL5 cDNA probe detected a major transcript of 3.8 kb in Burkitt's lymphoma cell lines and an aberrant transcript in the t(3;22) cell line, whereas no transcript was detected in myeloid, monocytoid, erythroid, T-lymphoid, and Epstein-Barr virus-immortalized B-lymphoblastoid cell lines.

Amino Acid Sequence↗

PubMed1994

Homozygosity mapping of the Werner syndrome locus (WRN).

Werner syndrome (WS) is an autosomal recessive disorder characterized by the early onset of several age-related diseases. The locus for this disease was recently mapped to 8p12. We studied 27 WS kindreds of mixed ethnic origins, 26 of which were consanguineous. In 24 of these families, the affected subject was given the diagnosis of "definite" WS and affected subjects in the remaining 3 pedigrees were given the diagnosis of "probable" WS. Affected subjects from each kindred were genotyped for 13 short tandem repeat polymorphic sites. Two-point linkage analysis yielded significant evidence for linkage to D8S137, D8S339, D8S87, PLAT, D8S165, and D8S166. The locus yielding a maximum lod score at the smallest recombination fraction was D8S339, suggesting that this marker is the closest to the WS gene (WRN locus) of those tested. D8S339 gave significant lod scores (Zmax > or = 3.0) for both Japanese and non-Japanese (mostly Caucasian) families, demonstrating that a single locus is responsible for WS in both groups. Multipoint analysis of these markers yielded a maximum lod score of 17.05 at a distance of approximately 0.6 cM from D8S339. The combined evidence from 2-point analysis, multipoint analysis, and analysis of regions of homozygosity in subjects from inbred pedigrees indicates that the WRN locus is between D8S131 and D8S87, in an 8.3-cM interval containing D8S339.

Alleles↗

PubMed1994

Characteristics of dynamic mutation in Japanese myotonic dystrophy.

To study the characteristics, if any, of unstable CTG repeat sequence in Japanese myotonic dystrophy (DM), we analyzed DNA from 351 at risk individuals (including affected and non-affected carriers and their descendants) from 105 families in Japan. A total of 93 DM families (196 affected and 116 unaffected individuals), including 84 DM parent-child pairs (44 father-child and 40 mother-child pairs), were examined, many of which had been previously tested by linkage analysis. We detected unstable CTG repeat mutations between 0.15 kb and 8.7 kb in size. The size of the mutation correlated with the age of onset of symptoms. There was a significant difference in DM allele size among the four groups (congenital, juvenile onset, classical, and minimal). Congenital DM had on average the largest repeat sizes. Comparison of parent-child pairs showed that most offspring had a larger repeat size than their parents, with only 2 of 84 showing a definite decrease in repeat size. The correlation coefficients for maternal and paternal transmission were 0.41 and 0.15, respectively. The parental age (maternal and paternal) did not correlate with intergenerational change of repeat. These observations are similar to those reported in Caucasians.

Adolescent↗