Biomedical subjects

T Kohda

Publications and source records attributed to T Kohda.

At least 37 records · Page 2Linked to original sources

PubMed1996

Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein.

Genetic and embryological studies in the mouse demonstrated functional differences between parental chromosomes during development. This is due to imprinted genes whose expression is dependent on their parental origin. In a recent systematic screen for imprinted genes, we detected Peg3 (paternally expressed gene 3). Peg3 is not expressed in parthenogenones. In interspecific hybrids, only the paternal copy of the gene is expressed in the embryos, individual tissues examined in d9.5-13.5 embryos, neonates and adults. Peg3 mRNA is a 9 kb transcript encoding an unusual zinc finger protein with eleven widely spaced C2H2 type motifs and two groups of amino acid repeats. Peg3 is expressed in early somites, branchial arches and other mesodermal tissues, as well as in the hypothalamus. Peg3 maps to the proximal region of chromosome 7. Consistent with our findings, maternal duplication of the proximal chromosome 7 causes neonatal lethality. This region is syntenic with human chromosome 19q13.1-13.3 (refs 10,11), where the genes for myotonic dystrophy and a putative tumour suppressor gene are located.

Amino Acid Sequence↗

PubMed1995

Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization.

Parthenogenesis in the mouse is embryonic lethal partly because of imprinted genes that are expressed only from the paternal genome. In a systematic screen using subtraction hybridization between cDNAs from normal and parthenogenetic embryos, we initially identified two apparently novel imprinted genes, Peg1 and Peg3. Peg1 (paternally expressed gene 1) or Mest, the first imprinted gene found on the mouse chromosome 6, may contribute to the lethality of parthenogenones and of embryos with a maternal duplication for the proximal chromosome 6. Peg1/Mest is widely expressed in mesodermal tissues and belongs to the alpha/beta hydrolase fold family. A similar approach with androgenones can be used to identify imprinted genes that are expressed from the maternal genome only.

Amino Acid Sequence↗

PubMed1995

Ribozymes: from mechanistic studies to applications in vivo.

The hammerhead ribozyme belongs to the class of molecules known as antisense RNAs. However, because of short extra sequences that form the so-called catalytic loop, it can act as an enzyme. Since the catalytic domain captures magnesium ions and magnesium ions can cleave phosphodiester bonds, hammerhead ribozymes are recognized as metalloenzymes. In general, the cleavage of phosphodiester bonds involves acid/base catalysis, with proton transfer occurring in the transition state. When the possibility of such a proton-transfer process was examined by measuring solvent isotope effects, it became apparent that no proton transfer occurs in the transition state during reactions catalyzed by a hammerhead ribozyme. It is likely, therefore, that hammerhead ribozymes exploit the general double-metal-ion mechanism of catalysis, with Mg2+ ions coordinating directly with the attacking and leaving oxygen moieties. Since the hammerhead ribozyme is one of the smallest RNA enzymes known and has potential as an antiviral agent, thus ribozyme has been extensively investigated for applications in vivo. Ribozymes are described that have possible utility as agents against HIV-1.

Animals↗

PubMed1995

Efficient replication of polyomavirus DNA in a cell-free system supplemented with Escherichia coli single-stranded DNA binding protein, which exhibits species-specificity in the requirement for DNA polymerase alpha-primase.

We established a modified cell-free system for polyomavirus (PyV) DNA replication, which was supplemented with Escherichia coli single-stranded DNA binding protein (SSB). DNA synthesis in this system was enhanced by 1.4- to over 15-fold depending upon the amount of cell extracts contained in the reaction mixture. By supplementing with E. coli SSB, we were able to reduce the amount of cell extracts in the reaction mixture, and to lower the concentrations of creatine phosphate and Tris, rendering this system more resistant to salts than the conventional PyV DNA replication system. The modified system was characterized using mutant cell extracts which had heat-inactivated DNA polymerase alpha. DNA synthesis in the system was dependent on PyV T antigen, the PyV origin of DNA replication, mutant cell extracts, and DNA polymerase alpha-primase complex purified from wild-type cells. The DNA polymerase alpha-primase complex was not replaced by DNA polymerase alpha, indicating that this system requires a functional DNA polymerase alpha-primase complex. This system exhibited species-specificity in the requirement for DNA polymerase alpha-primase; only mouse DNA polymerase alpha-primase but not human DNA polymerase alpha-primase functioned in this system.

Animals↗

PubMed1995

Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Animals↗

PubMed1994

Characterization and transcriptional regulation of rat glutamine tRNA-encoding genes: repression of tRNA(UUGGln) synthesis.

In rat liver, the amount of tRNA(UUGGln) (RG-2) was found to be approx. 20% of that of tRNA(CUGGln) (RG-1). The independent RG-1 and RG-2 genes were isolated from a rat genomic library together with four RG-related genes containing two to five alterations in their coding regions. Sequence analysis demonstrated that there was no difference between the internal promoter sequences of RG-1 and RG-2. However, interestingly, the transcriptional activity of RG-1 was approximately four-times higher than that of RG-2 in an in vitro transcription reaction. Replacement of the 5'-flanking sequence of RG-2 by the corresponding sequence of RG-1 or by a plasmid DNA sequence caused activation of RG-2 transcription. Gel retardation assay demonstrated that the 5'-flanking region of RG-2 contained a unique sequence specifically recognized by a nuclear protein. Taken together, these results strongly suggest that the transcriptional activity of RG-2 might be negatively regulated by the binding of a nuclear protein at a specific site in the 5'-flanking region of the gene.

Animals↗

PubMed1994

Purification of two DNA-dependent adenosinetriphosphatases having DNA helicase activity from HeLa cells and comparison of the properties of the two enzymes.

DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks designated Q1 to Q5 by FPLC Mono Q column chromatography. In our previous study, we observed that crude extracts prepared from xeroderma pigmentosum complementation group C (XP-C) cells contained no DNA-dependent ATPase activity at the peak position of Q1 and exhibited a broader peak with higher activity than normal Q2 at the peak position of Q2 [Yanagisawa, J., Seki, M., Ui, M., & Enomoto, T. (1992) J. Biol. Chem. 267, 3585-3588]. We have purified two DNA-dependent ATPases Q1 and Q2 from HeLa cells and characterized their properties in order to obtain a means to discriminate ATPase Q1 from Q2 in XP-C cells. The apparent molecular masses of Q1 and Q2 on SDS-polyacrylamide gel electrophoresis were 73 and 100 kDa, respectively. The two enzymes required a divalent cation for activity. DNA-dependent ATPase Q1 hydrolyzed ATP and dATP and Q2 hydrolyzed ATP preferentially among the nucleotides tested. Both enzymes preferred single-stranded DNA as a cofactor. The DNA-dependent ATPase activity of Q2 was inhibited by 90% in the presence of 200 mM NaCl, whereas that of Q1 was not affected by NaCl at concentrations up to 200 mM. Both enzymes had DNA helicase activity, that of Q1 being more resistant to NaCl than that of Q2. The DNA helicase activity of Q2 was about 150-fold higher than that of Q1, when compared with units of ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗

PubMed1992

DNA-dependent adenosinetriphosphatase C1 from mouse FM3A cells has DNA helicase activity.

In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).

Adenosine Triphosphatases↗

PubMed1992

Production of an immunoglobulin gene product by the plasmid expression vector L factor in mouse myeloma cells.

L factor, originally discovered in a subclone of mouse L cells, is a multicopy mammalian plasmid whose structure is related to that of polyoma. When a composite DNA consisting of L factor, pBR, bacterial neo, and an immunoglobulin (kappa) gene was introduced into mouse myeloma cells, the DNA was established as plasmids in the cells without rearrangement or integration into the chromosomes. The plasmid-bearing myeloma cells produced kappa mRNA and the gene product, kappa immunoglobulin, which were apparently derived from the gene located on plasmid L factor. These results suggest that L factor can be used as a plasmid expression vector for studies on gene expression and production of biologically active substances in mammalian cells.

Animals↗

PubMed1991

Cellular HSP90 (HSP86) mRNA level and in vitro differentiation of mouse embryonal carcinoma (F9) cells.

Treatment of mouse embryonal carcinoma (F9) cells with retinoic acid, an inducer of F9 cell differentiation, greatly increased the level of mRNA specific to one of the heat-shock proteins (HSP86). Experiments including the one employing differentiation-resistant mutant F9 cells suggested that the increase represents early molecular events associated with the embryonal differentiation. The increased HSP86 mRNA declined to the original level during further incubation. The presence of cyclic AMP, which stimulates conversion of the retinoic acid-induced primitive endoderm cells to parietal endoderm cells, prevented the decline. These results suggest that not only the elevation of HSP86 mRNA level represents early molecular events in F9 cell differentiation but also that sustaining the elevated level (by cyclic AMP) is associated with further differentiation of the embryonal cells.

Animals↗

PubMed1991

Structural and functional analysis of a polyoma-related mammalian plasmid (L factor): the enhancer activity and plasmid establishment.

L factor is a unique plasmid DNA which was originally discovered in a subclone (B822) of mouse L cells at a high copy number (more than 5,000 copies/cell). The presence of L factor caused no detectable abnormalities to the plasmid-bearing cells. We determined the total DNA sequence of the L factor I (and a part of L factor II) and compared it with that of polyoma DNA. Both DNA are common to the general construction of DNA frames such as early, late and noncoding regions, suggesting the two to be closely related. On the other hand, the L factor DNA sequences differ substantially from that of polyoma in the DNA sequences corresponding to the polyoma large T antigen, capsid proteins and a portion of the enhancer region. In order to investigate the mechanism of plasmid establishment of L factor, we compared the enhancer activity, capacity of DNA replication and efficiency of plasmid establishment of L factor with those of polyoma. The results indicate that L factor enhancer activity and DNA replication capacity were considerably lower than those of polyoma, suggesting that these altered (lowered) activities associated with L factor contribute to the plasmidal establishment and stable maintenance of L factor.

Animals↗

PubMed1991

Induction of CAT gene expression on a plasmid vector (L factor) by retinoic acid in mouse embryonal carcinoma (F9) cells.

We reported previously that composite DNA constructed from a mammalian plasmid (L factor) and foreign gene can be reestablished as a plasmid in mouse embryonal carcinoma (F9) cells after transfection and the plasmid-bearing F9 cells undergo normal in vitro differentiation in response to retinoic acid, an inducer for F9 cell differentiation. We constructed F9 cells bearing plasmidal L factor DNA in which a reporter (chloramphenicol acetyltransferase; CAT) gene was placed under the control of a differentiation-responsive viral (Moloney murine leukemia virus or simian virus 40) enhancer-promoter. When such plasmid-bearing cells were treated with retinoic acid, the CAT gene was inducibly expressed. These results indicate that mammalian gene expression can be studied with the plasmidal expression vector which is structurally dissociated from complex chromosomes.

Animals↗

PubMed1991

Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level.

Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed.

Benzamides↗

PubMed1989

Two new carbapenem antibiotic-producing actinomycetes: Kitasatosporia papulosa sp. nov. and Kitasatosporia grisea sp. nov.

Two new carbapenem antibiotic-producing actinomycetes, the cell-walls of which contain LL-diaminopimelic acid and meso-diaminopimelic acid, were isolated from soil. The two strains were subjected to taxonomic studies, which involved morphological, cultural, physiological and chemotaxonomical characterization, the latter including the cell-wall chemo-type, whole-cell sugar composition, phospholipid composition, menaquinone system and DNA base composition. These strains were identified as new species of the genus Kitasatosporia. The proposed names are Kitasatosporia papulosa for strain AB-110 (IAM 13637, FERM 9000, JCM 7250) and Kitasatosporia grisea for strain AA-107 (IAM 13638, JCM 7249).

Actinomycetales↗

PubMed1989

Highly targeted screening system for carbapenem antibiotics.

An improved screening system in a search for carbapenem antibiotics from actinomycetes was developed, involving (1) effective isolation of carbapenem antibiotic producers, (2) favorable cultivation as to antibiotic production and (3) simplified identification of the carbapenem antibiotics: (1) A frequency of isolation of carbapenem antibiotic producers from soils was increased by the use of an amoxicillin/clavulanate-containing medium. As many as 20% of the actinomycetes isolated in this medium were found to be carbapenem antibiotic producers, as compared with 0.6% when conventional media were employed. (2) An agar-surface cultivation on oatmeal - yeast extract - malt extract (OMYM) medium, containing oatmeal, yeast extract, malt extract, glucose and trace elements, was most suitable for the antibiotic production. (3) An agar-diffusion assay for identification of carbapenem antibiotics was developed. Although the carbapenem antibiotics are stable against beta-lactamases, they were hydrolyzed completely with excess amounts of common beta-lactamases. So they could be discriminated from other beta-lactam antibiotics and non-beta-lactam antibiotics by comparison of the susceptibility profiles for such enzymes.

Actinomycetales↗

PubMed1989

Identification of bile alcohols in rat bile.

Bile alcohols in rat bile were analyzed by gas-liquid chromatography-mass spectrometry. Six bile alcohols were newly identified as minor constituents in addition to 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, major bile alcohol of rat bile. The bile alcohols newly identified were 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, and 5 beta-cholestane-3 alpha,6 beta,7 beta,25,26-pentol. The biliary bile alcohols of the rat occurred mainly as the sulfuric acid esters and, in lesser amounts, as glucuronoconjugated and unconjugated forms. The amount of total bile alcohols was about 27.9 nmol in 1 ml of bile.

Animals↗

PubMed1988

Establishment of composite DNA derived from L factor as a plasmid in mouse embryonal carcinoma (F9) cells.

We have recently reported a mammalian cell plasmid (L factor) whose structure is related to that of polyomavirus (T. Kusano, H. Uehara, H. Saito, K. Segawa, and M. Oishi, Proc. Natl. Acad. Sci. USA 84:1789-1793, 1987). When composite DNA constructed from L factor and a foreign gene was introduced into mouse embryonal carcinoma (F9) cells by transfection, the DNA was reestablished in the cells as a plasmid. The reestablished plasmid DNA in F9 cells could be rescued in Escherichia coli. The plasmid-bearing cells underwent normal in vitro differentiation in response to retinoic acid. The efficiency of plasmid establishment of the L-factor-derived DNA and transcriptional and transient replicational activities were compared with those of similar composite DNA constructed from polyomavirus and an embryonal carcinoma mutant of polyomavirus which is permissive in F9 cells. The results suggest an inverse relationship between the efficiency of the plasmid establishment and the activity of gene expression controlled by the intrinsic enhancer-promoter of the DNA.

Animals↗

PubMed1988

Measurement of serum bile alcohol levels in liver dysfunction, using isotope dilution-mass spectrometry.

We determined the concentration of two major serum bile alcohols, 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, in healthy controls and patients with acute hepatitis, chronic hepatitis, and cirrhosis, using isotope dilution-mass spectrometry. In healthy controls the mean total amount of the two major bile alcohols was 9.6 +/- 3.4 ng/ml (mean +/- SD). In patients with liver diseases the mean values were as follows: acute hepatitis, 44.8 +/- 18.2 ng/ml; chronic hepatitis, 18.2 +/- 5.6 ng/ml; Child grade A cirrhosis, 37.9 +/- 26.0 ng/ml; Child grade B, 42.7 +/- 22.0 ng/ml; and Child grade C, 71.2 +/- 24.2 ng/ml. The mean ratio of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol to 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol in patients with Child grade C cirrhosis was significantly higher than that in healthy controls (1.42 +/- 0.92 versus 0.61 +/- 0.11; p less than 0.05).

Adult↗