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Biomedical subjects

J Kuwahara

Publications and source records attributed to J Kuwahara.

At least 19 recordsLinked to original sources
PubMed2001

Involvement of PLAGL2 in activation of iron deficient- and hypoxia-induced gene expression in mouse cell lines.

We searched iron-deficient inducible cDNA, using subtraction cloning and mRNA from desferrioxamine-treated mouse macrophage Raw264.7 cells. We identified a pleomorphic adenoma gene like 2 (PLAGL2), one of PLAG superfamily proteins exhibiting antiproliferative properties on tumor cells. Mouse PLAGL2 consists of 496 amino acids with seven C2H2 zinc-fingers. PLAGL2 mRNA was induced in RAW264.7 cells, mouse erythroleukemia cells and Balb/c 3T3 cells when they were treated with desferrioxamine. Hypoxia also increased PLAGL2 mRNA. Expression of PLAGL2 in COS-7 cells led to nuclear localization. PLAGL2 had potential binding ability to GC-rich oligonucleotide and activated transcription of a gene with the binding sequence in transient reporter assay, a finding consistent with a case seen in a PLAGL2 homolog, ZAC-1. Transient co-transfection of PLAGL2 or ZAC1 cDNA and a reporter containing a lactate dehydrogenase A (LDHA) promoter carrying the hypoxia inducible factor-1 responsive element led to an increase in the basal transcription in Balb/c 3T3 and HepG2 cells. Activation in transcription from the LDHA promoter increased by desferrioxamine treatment or hypoxia was further enhanced when PLAGL2 was expressed. We propose that PLAGL2 is involved in the cell cycle arrest and apoptosis of tumor cells by regulating iron depletion- or hypoxia-inducible gene expression.

Amino Acid Sequence↗
PubMed2000

Zn finger and nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is located in the nucleus of a mammalian cell and importantly related to expression of many cellular genes. In order to elucidate the nuclear localization mechanism of Sp1, various truncated fragments of Sp1 were fused to green fluorescent protein (GFP) and expressed in HeLa cell. The results show significance of the DNA binding region, especially, zinc finger (Zn finger) domain for nuclear localization of Sp1 in HeLa cell.

Binding Sites↗
PubMed1999

Nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is importantly related to expression of many cellular genes. In order to identify the nuclear localization signal of Sp1, various truncated regions of Sp1 were fused to green fluorescent protein (GFP) and were expressed in HeLa cell. The C-terminal region are required to localize Sp1 in the nucleus of HeLa cell.

Cell Nucleus↗
PubMed1996

Gene of rat Ca2+/calmodulin-dependent protein kinase II alpha isoform -- its cloning and whole structure.

The gene encoding the alpha isoform of rat Ca2+/calmodulin-dependent protein kinase II was cloned, and its exon-intron organization was analyzed. The coding region of cDNA consists of 18 exons spanning more than 50 kilobase pairs. Each of the discrete functional units, such as the ATP-binding site, the autophosphorylation site responsible for Ca2+-independent activity, the calmodulin-binding site, and link structure is encoded by a single exon. The largest and smallest exons consist of 229 and 41 base pairs, respectively. All splice junction sequences flanking the introns conform to the consensus splice junction sequence and the GT-AG splice rule.

Adenosine Triphosphate↗
PubMed1993

Alteration of DNA binding specificity by nickel (II) substitution in three zinc (II) fingers of transcription factor Sp1.

Transcription factor Sp1, which has a DNA binding domain composed of three zinc fingers, binds to GC box (consensus sequence, G/T-GGGCGG-G/A-G/A-C/T) and activates the transcription by RNA polymerase II. Metal substitution of nickel(II) for zinc(II) in Sp1 causes no differences in the mode of protein-DNA interaction. However, sequence preference of Ni(II)Sp1 changes from 5'-GGGGCGGGGC to 5'-GGGGCGTGGC, and is distinct from that of Zn(II)Sp1. The result indicates an important effect of metal-induced folding on sequence-specific recognition of DNA by zinc-finger proteins.

Base Sequence↗
PubMed1993

Guanine-specific binding by bleomycin-nickel(III) complex and its reactivity for guanine-quartet telomeric DNA.

Bleomycin-Ni(III) [BLM-Ni(III)] complex, generated from oxidation of the corresponding BLM-Ni(II) complex with Ir(IV) or oxone, binds to single-stranded DNA and causes strand scission at specific guanine sites. In the telomeric-like oligodeoxynucleotide d(T2G4)4, the cleavage by the BLM-Ni(III) complex occurs preferentially at positions G-9 and G-15 in loop regions. By contrast, this Ni(III) complex does not induce cleavage of G-quartet d(G4T4G4)2, which contains no loop guanines, ESR evidence supports that binding of the N-7 position of guanine occurs axially to the Ni(III) center in BLM. The present results provide interesting insights into Ni-BLM chemistry.

Base Sequence↗
PubMed1993

Binding of transcription factor Sp1 to GC box DNA revealed by footprinting analysis: different contact of three zinc fingers and sequence recognition mode.

Transcription factor Sp1 has three tandem repeats of a Cys2His2-type zinc finger motif and specifically binds to GC box DNA. We investigated the interaction of Sp1 with GC box DNA by several footprinting techniques. Methylation of four guanine bases in the sequence 5'-GGGCG-3' is strongly protected by Sp1 binding, whereas a guanine base flanked at the 5' end by the above sequence is extremely hypermethylated. Methylation interference experiments explicitly show that four guanine bases from the guanine-rich strand, and one from the cytosine-rich strand, in the sequence 5'-GGGCG-3' are crucially required for GC box recognition by Sp1. In footprinting using the 1,10-phenanthroline-copper complex, binding of Sp1 clearly alters the cleavage patterns by the metal complex. Footprints of the protein did not cover the full length of each GC box sequence, and the protein strongly masked scission in the sequence 5'-GCGG(A/G)(G/A)-3'. In cleavage of GC box DNA by the bleomycin-iron complex, Sp1 binding induces new cutting at a 5'-GA-3' site within the box. The results indicate that (i) the three zinc fingers do not contribute equivalently to the binding of Sp1 to the GC box, namely, important base contacts arise from the second and third fingers, and (ii) the protein binding induces local but significant structural distortion of the 3' region of the guanine-rich strand in the GC box. These features are clearly distinct from those of Zif268 and Krox20, which are three-zinc-finger proteins closely related to Sp1.

Amino Acid Sequence↗
PubMed1993

Cleavage of yeast tRNA(phe) with Ni(III) and Co(III) complexes of bleomycin.

The BLM-Ni(III) complex preferentially cleaves guanine residues at D loop and anticodon loop of yeast tRNA(phe) by aniline treatment, whereas the BLM-Co(III) complex degrades certain 5'-AN sites of the minor groove region after irradiation of UV light. These cleavage features are clearly different from that of the BLM-Fe(II) complex.

Base Sequence↗
PubMed1993

DNA binding and cleavage of a novel antitumor antibiotic dynemicin A: detection of binding and cleavage sites by exonuclease III digestion and alkali-induced cutting reactions.

We found here that dynemicin A effectively breaks DNA strands under alkaline pH condition. Binding of dynemicin A to double-stranded DNA clearly interrupts the digestion reaction of exonuclease III. The DNA association sites of dynemicin A correspond considerably well to its cleavage sites. Dynemicin A seems to intercalate preferentially into the relaxed region of the DNA double helix. On the other hand, the alkali-product of dynemicin A was chromatographically identified with dynemicin N, suggesting a DNA cleavage mechanism similar to the reductant- and light-induced activation systems of dynemicin A. In order to detect drug binding to the relaxation structure in the DNA duplex, the present exonuclease III-digestion-stop-sequence method is useful.

Anthraquinones↗
PubMed1992

Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action.

In view of the cationic amphipathic structure of tachyplesin I and antiparallel beta-sheet as a general DNA binding motif, DNA binding of the antimicrobial peptide has been examined. Several footprinting-like techniques using DNase I protection, dimethyl sulfate protection, and bleomycin- (BLM-) induced DNA cleavage were applied in this study. Some distinct footprints with DNase I are detected, and also the sequence-specific cleavage mode of the BLM-Fe(II) complex clearly is altered in the presence of tachyplesin I. In addition, methylation of the N-7 residue of guanine situated in the DNA major groove is not entirely inhibited (or activated) by tachyplesin I. The results suggest that tachyplesin I interacts with the minor groove of DNA duplex. Disappearance of the footprints by dithiothreitol-treated tachyplesin I and Ala-tachyplesin strongly suggests a significant contribution of secondary structure containing an antiparallel beta-sheet to the DNA binding of tachyplesin I. This is the first report on DNA interaction with a small peptide which contains a unique antiparallel beta-sheet structure. The mechanism for antimicrobial action of tachyplesin I has also been inferred.

Amino Acid Sequence↗
PubMed1992

Recognition and structural perturbation of GC box DNA by Sp1 zinc finger.

Interaction of Sp1 with GC box DNA was investigated by several footprinting experiments. Methylation of four guanine bases is strongly protected by Sp1 binding, while one guanine base in GC box is extremely hypermethylated. Sp1 binding also induces new cleavage at 5'-GA-3' site within GC box by bleomycin-iron complex.

Amino Acid Sequence↗
PubMed1991

Metal binding and DNA recognition in transcription factor Sp1.

DNA binding domain of Sp1 encompassing three Cys2His2-type Zn-finger motifs is cloned and expressed in E.coli. The Sp1 fragment shows metal-dependent folding and DNA binding. The Zn(II)-induced folding of the three fingers is probably cooperative. Release of one equivalent of Zn decreases but does not abolish DNA binding activity of Sp1.

Amino Acid Sequence↗
PubMed1990

Role of the zinc(II) ions in the structure of the three-finger DNA binding domain of the Sp1 transcription factor.

The transcription factor Sp1 from Hela cells contains near the C-terminus of this protein of 778 amino acids three contiguous repeats of an amino acid sequence, -Cys-X4-Cys-X12-His-X3-His-, typical of the Cys2His2-type zinc-finger DNA binding domain first found in transcription factor TFIIIA. A DNA sequence corresponding to the condons from residue 614 to residue 778 of Spl (encompassing the three zinc-finger motifs) has been cloned and overproduced in Escherichia coli. The fragment of Sp1 containing the C-terminal 165 residues plus 2 from the cloning vector, designated Sp1(167*), can be extracted with 5 M urea and then refolded in the presence of Zn(II) to a protein of specific conformation containing 3.0 +/- 0.2 mol of tightly bound Zn(II)/mol of protein. Gel retardation assays using a labeled 14-bp DNA sequence containing a consensus Sp1 binding site show that the refolded Zn(II) protein specifically recognizes the "GC box" sequence in the presence of a large excess of calf thymus DNA. Treatment of Zn(II)Sp1(167*) with 10 mM EDTA results in removal of Zn(II) and the formation of an apoprotein which does not specifically recognize DNA. Cd(II) can be exchanged for Zn(II) in the refolded protein with full retention of specific DNA recognition. This is the first Cys2His2-type "finger" protein where this substitution has been accomplished. Titration of the Zn(II) protein with 6 mol of p-mercuribenzenesulfonate/mol of protein results in the complete release of the three Zn(II) ions. Release of Zn(II) is highly cooperative.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

Light-induced DNA cleavage by esperamicin and neocarzinostatin.

Ultraviolet radiation of the enediyne drugs is effective in causing nicks in supercoiled DNA. Of special interest is the fact that the observed nucleotide cleaving specificity for the UV light- and thiol-activated antibiotics was the same with esperamicin A1, but was different with neocarzinostatin. In addition to the preferred cutting of T and A bases, the light-activated neocarzinostatin attacked certain G bases which were rarely cleaved by the thiol-activated neocarzinostatin. It should be noted that these enediyne antibiotics lose the DNA breakage activity after light-exposure for 30 min.

Aminoglycosides↗
PubMed1989

Nucleotide-specific cleavage and minor-groove interaction of DNA with esperamicin antitumor antibiotics.

The cleavage of DNA by esperamicin is greatly accelerated in the presence of thiol compounds. Oxygen and active oxygen-radical scavengers have no significant influence upon DNA strand breakage by esperamicin. The preferential cutting sites of esperamicin are at thymidylate residues, and the frequency of bases attacked (T greater than C greater than A greater than G) is different from that of calicheamicin (C much greater than T greater than A = G), neocarzinostatin (T greater than A greater than C greater than G), or bleomycin (C greater than T greater than A greater than G). Esperamicin preferentially attacks at T and C bases in oligopyrimidine sequences such as 5'-CTC-3', 5'-TTC-3', and 5'-TTT-3'. In contrast to the preferred sites of cleavage by bleomycin, 5'-GT-3' and 5'-GC-3', the preferred sites of esperamicin-mediated DNA degradation are 5'-TG-3' and 5'-CG-3' sequences. The nucleotide-specific cleavage mode of esperamicin is significantly affected by pretreatment of DNA with netropsin and distamycin A, suggesting that interaction of esperamicin occurs through the minor groove of B-DNA. This is further supported by the asymmetric cleavage pattern to the 3' side on the opposite strand of the DNA. The roles of the fucose-anthranilate moiety and the trisaccharide side chain of esperamicin in DNA binding and base recognition are discussed.

Aminoglycosides↗
PubMed1988

Significant interaction between low-spin iron(III) site and pyrroloquinoline quinone in active center of nitrile hydratase.

Nitrile hydratase, a unique non-heme iron enzyme, contains low-spin Fe(III) site and pyrroloquinoline quinone in the active center. The enzymatic activity of nitrile hydratase is strongly inhibited by typical carbonyl reagents such as phenylhydrazine and semicarbazide. Of special interest is the fact that these carbonyl reagents induced significant alteration of the ESR features of the native iron(III) enzyme at pH 7.2. In addition, the isobutyronitrile(inhibitor)-bound enzyme was also remarkably affected by phenylhydrazine and then the transformed ESR characteristics were different from those of phenylhydrazine-treated enzyme. The present results reveal the first evidence for an important interaction between the low-spin Fe(III) site and pyrroloquinoline quinone which is essential for the activity of nitrile hydratase.

Binding Sites↗
PubMed1988

Sequence-specific recognition and cleavage of DNA by metallobleomycin: minor groove binding and possible interaction mode.

The DNase I cleavage-inhibition analysis shows binding sites of approximately 2 or 3 base pairs--in particular, 5' N-G-C sequences--for the green-colored CoIII and fully oxidized FeIII complexes of bleomycin. The apparent binding constant of the bleomycin-CoIII complex is quite similar for glucosylated and nonglucosylated phage T4 DNAs, whereas poly[d(I-C)] clearly gives a smaller binding constant than does poly[d(G-C)]. In contrast to the covalent attachment of the guanine N-7 with aflatoxin B1, the modification of the guanine 2-amino group with anthramycin remarkable inhibits the DNA cleavages at 5' G-C and 5' G-T sites by the FeIII and CoIII complex systems of bleomycin. These results strongly indicate that metallobleomycin binds in the minor groove of B-DNA and that the 2-amino group of guanine adjacent to the 5' side of the cleaved pyrimidine base is one key element of the specific 5' G-C or G-T recognition by the bleomycin-metal complex. A possible binding mode of metallobleomycin in the DNA helix has been proposed by computer-constructed model building.

Base Sequence↗