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K Hitomi

Publications and source records attributed to K Hitomi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cloning and characterization of a gene (UVR3) required for photorepair of 6-4 photoproducts in Arabidopsis thaliana.

UV radiation induces two major classes of pyrimidine dimers: the pyrimidine [6-4] pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimer (CPD). Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. A gene that expresses a protein with 6-4 photolyase activity in vitro was recently cloned from Drosophila melanogaster and Xenopus laevis. We report here the isolation of a homolog of this gene, cloned on the basis of sequence similarity, from the higher plant Arabidopsis thaliana. This cloned gene produces a protein with 6-4 photolyase activity when expressed in Escherichia coli. We also find that a previously described mutant of Arabidopsis (uvr3) that is defective in photoreactivation of 6-4 products carries a nonsense mutation in this 6-4 photolyase homolog. We have therefore termed this gene UVR3. Although homologs of this gene have previously been shown to produce a functional 6-4 photolyase when expressed in heterologous systems, this is the first demonstration of a requirement for this gene for photoreactivation of 6-4 products in vivo.

Amino Acid Sequence↗
PubMed1998

Ascorbate-dependent enhancement of nitric oxide formation in activated macrophages.

Macrophages activated with bacterial lipopolysaccharide (LPS) and cytokines produce nitric oxide through the induction of iNOS gene expression. Ascorbate increased NOx (nitrite and nitrate) formation by approximately 40% in a mouse macrophage-like cell line, J774.1, activated with LPS and interferon-gamma. Ascorbate alone exhibited no inductive activity toward NO formation. N(G)-Monomethyl-L-arginine inhibited nitrite formation in cells activated in the presence or absence of ascorbate. Northern and Western blotting analyses showed that both iNOS mRNA and protein steady-state levels were increased approximately twofold in cells activated in the presence of ascorbate compared to in cells activated only with the inducers. These data suggest that ascorbate increased NO production by increasing the amount of iNOS in the activated macrophages.

Animals↗
PubMed1998

Expression of biologically active human calpastatin in baculovirus-infected insect cells and in Escherichia coli.

Calpastatin, an endogeneous inhibitor protein acting on calpain (Ca(2+)-dependent cysteine proteinase), is widely distributed in animal tissues and cells. Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Escherichia coli, were used for overexpression of the human calpastatin tagged with N-terminal hexahistidine peptide. Recombinant calpastatin was purified to homogeneity by nickel ion affinity chromatography and gel filtration separation. Purified recombinant proteins from both systems have similar inhibitory activity for calpain.

Amino Acid Sequence↗
PubMed1997

Binding and catalytic properties of Xenopus (6-4) photolyase.

Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6-4]T in double-stranded DNA with high affinity (KD = 10(-9)) and to T[6-4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (KD = approximately 2 x 10(-8)). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (koff = 2.9 x 10(-5) s-1). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (phi) of repair is approximately 0.11. The fully reduced form (E-FADH-) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis, syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.

Animals↗
PubMed1997

Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit.

The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.

Animals↗
PubMed1997

Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.

Animals↗
PubMed1997

Characterization of a human homolog of (6-4) photolyase.

(6-4)Photolyase catalyzes light-dependent repair of UV-induced pyrimidine (6-4) pyrimidone photoproducts. A human cDNA clone which has high sequence homology to the (6-4)photolyase gene (H64PRH gene) was identified. In this paper we also isolated a genomic clone corresponding to the H64PRH cDNA and mapped it to chromosome 12q24.1 by fluorescence in situ hybridization (FISH). Northern-blot analysis revealed transcription of this gene in all human tissues examined. The H64PRH protein was overproduced in E. coli, partially purified and characterized. Like (6-4)photolyase, the enzyme contains two chromophores, one of which is FAD. However, the enzyme does not show any detectable photolyase activity.

Chromosome Mapping↗
PubMed1997

Ascorbate-dependent expression of ubiquitin genes in guinea pigs.

In an attempt to characterize a gene(s), of which the expression is ascorbate-dependent, a cDNA fragment encoding ubiquitin was isolated from a subtracted cDNA library constructed from spleen RNAs of ascorbate-deficient or -replete guinea pigs. On Northern blot analysis, three transcripts (1.8 kb ubiX, 1.3 kb ubiY and 0.7 kb ubiZ) were detected. The ubiY encodes four direct repeats of the 76 amino acid ubiquitin sequence with seven additional amino acids, V-Y-A-S-P-I-F, at the C-terminus. The transcript ubiX appears to comprise more than five repeats of the ubiquitin-encoding unit. The ubiZ encodes a ubiquitin monomer fused to an 80 amino acid extension exhibiting 100% amino acid sequence identity to the human homolog, HUMUBA80R. The ubiX gene was expressed animal-dependently. The ubiY mRNA levels decreased under ascorbate-deficient conditions, and increased under ascorbate-replete conditions, whereas ubiZ mRNA remained unaltered at low levels under the feeding conditions used here.

Amino Acid Sequence↗
PubMed1996

Synergistic effect of BMP-2 and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells.

Of TGF-beta superfamily proteins, BMP-2 enhanced alkaline phosphatase (ALP) activity in cultured osteoblastic cells, MC3T3-E1, to the same level promoted by ascorbate, whereas TGF-beta s (beta 1, beta 2, beta 3) reduced ALP activity and altered cell morphology under the same conditions. Activin appeared to have no distinct effect on ALP synthesis. Ascorbate dependent increase in ALP synthesis was markedly stimulated in the presence of BMP-2. The synergistic effect of ascorbate on ALP synthesis was replaced by type I collagen coated on the culture dish. However, BMP-2 appeared not to bind to type I collagen. These findings indicate that BMP-2 acts directly on osteoblastic cells through its receptors and collagenous matrix can neither recruit BMP-2 nor modulate directly the action of BMP-2 in the pericellular area. Treatment of cells grown in ascorbate media with TGF-beta s decreased rapidly the cellular ALP activity indicating that TGF-beta s direct cells to the dedifferentiated stage.

Activins↗
PubMed1996

4-Hydroxy-2-nonenal cytotoxicity in renal proximal tubular cells: protein modification and redox alteration.

4-Hydroxy-2-nonenal (HNE), one of the major products of membrane lipid peroxidation, has been shown recently to be present in a form covalently attached to proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate (Toyokuni, S., et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620; Uchida, K., et al. (1995) Arch. Biochem. Biophys. 317, 405-411). In the present study, the mechanism of HNE cytotoxicity was studied using the renal tubular epithelial cells (LLC-PK1), focusing on the protein modification and alteration of cellular redox status induced by HNE. Upon treatment with HNE for 2 h, the LLC-PK1 cells were found to be resistant to the low concentration (10 microM) of HNE, while HNE at higher concentrations (> or = 50 microM) mediated cell death. The cytotoxicity of HNE appeared to be correlated with the HNE modification of cellular proteins. Among a number of proteins modified by HNE, a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was detected as one of the major targets of HNE in the cells. On the other hand, exposure of LLC-PK1 cells to HNE resulted in rapid reduction of cellular glutathione (GSH) levels, suggesting that HNE influenced primarily the redox status of the cells. Depletion of GSH with buthionine sulfoximine, a potent suppressor of GSH biosynthesis, before HNE treatment caused the cells to be sensitive to HNE cytotoxicity and to HNE modification of cellular proteins, whereas the increase in intracellular GSH levels by treatment with N-acetylcysteine before HNE treatment resulted in a dose-dependent inhibition of HNE-mediated protein modification. These results suggest that intracellular GSH is a determinant on cellular resistance against the HNE-mediated cytotoxicity.

Acetylcysteine↗
PubMed1996

Demonstration of alkaline phosphatase participation in the mineralization of osteoblasts by antisense RNA approach.

MC3T3-E1 cells grown with ascorbic acid express sequentially osteoblastic marker proteins such as alkaline phosphatase (ALPase) and then form a mineralized extracellular matrix (ECM) as a consequence of osteoblastic differentiation. To explore the functional roles of ALPase in the process of osteoblastic maturation, an inducible expression vector for antisense ALPase RNA was constructed and stably transfected into MC3T3-E1 cells. The expression of antisense ALPase RNA in the differentiated MC3T3-E1 transfectants reduced markedly the ALPase activity, which resulted in a significant decrease in the deposition of minerals upon prolonged culture. These findings demonstrated directly that ALPase participated in the mineralization of ECM.

Alkaline Phosphatase↗
PubMed1996

Enhancement effect of O6-fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells.

O6-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O6-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O6-fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O6-benzylguanine and O6-phenylguanine. The O6-(4- and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O6-benzylguanine. In contrast, O6-(2-fluorobenzyl)guanine and O6-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O6-(4- and 3-fluorobenzyl) guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O6-(2-fluorobenzyl)guanine and O6-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O6-fluorobenzylguanine derivatives. O6-Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O6-benzylguanine and were powerful MGMT inactivators. The chemical features of the O6-benzyl group are a biologically important determinant in the reaction evolution with MGMT.

Antineoplastic Agents↗
PubMed1995

[Neonatal apnea after general anesthesia--effects of intraoperative hyperventilation and serum ionized Ca concentration].

The relationship between neonatal apnea following general anesthesia and serum ionized calcium (Ca2+) concentration was examined in 13 neonates who had received intraoperative hyperventilation. In all cases, preoperative serum Ca2+ concentration was within normal limits. The anesthesia was maintained by nitrous oxide and oxygen. At the end of anesthesia, the incidence of abnormal breathing such as apnea, periodic breathing or subcostal retraction and their activity were investigated. Five minutes after intravenous administration of 2% CaCl2 solution (16 mg.kg-1), the same parameters were compared with the values before CaCl2 administration. As we used only Ca2+ free solution for fluid therapy during operation, serum Ca2+ concentration decreased gradually under general anesthesia, but after CaCl2 administration, it increased and the incidence of abnormal breathing decreased. To determine the relationship between hyperventilation and the incidence of abnormal breathing, the data were analyzed by dividing the patients into two groups based on PaCO2 level, a lower PaCO2 group (intraoperative PaCO2 < 30 mmHg, n = 5) and a higher PaCO2 group (PaCO2 > or = 30 mmHg, n = 8). But there was no significant relationship between them. In conclusion, this study demonstrates that the Ca administration has a favorable effect on respiratory system and motor activity, but we cannot relate the incidence of postoperative abnormal breathing to the degree of hyperventilation.

Anesthesia, General↗
PubMed1994

cDNA sequence for rkST1, a novel member of the sodium ion-dependent glucose cotransporter family.

A full length cDNA for rkST1, a novel member of the Na+/glucose cotransporter family, was cloned from rabbit kidney and sequenced. The coding sequence comprised 2022 base pairs and 674 amino acids. rkST1 beared 50-60% amino acid identity to the other cotransporters and was characteristic in respect of its expression in brain in addition to kidney among the cotransporters.

Amino Acid Sequence↗