Biomedical subjects

S Tanuma

Publications and source records attributed to S Tanuma.

At least 37 records · Page 2Linked to original sources

PubMed1998

Involvement of cytosolic NAD+ glycohydrolase in cyclic ADP-ribose metabolism.

The NAD+ glycohydrolase homogeneously purified from bovine brain cytosol was found to catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). Although the formation of cADPR from NAD+ does not exceed about 2% of the reaction products, the cyclase activity is clearly evidenced by its conversion of NGD+ to cyclic GDP-ribose (cGDPR), which cannot be hydrolyzed to GDPR. Importantly, a steep increase in cADPR hydrolytic activity was observed at cADPR concentrations above 60 microM, which could be reproduced on a Hill curve with a Hill coefficient of 2. Thus, the allosteric binding of cADPR to the NAD+ glycohydrolase (E) molecule promotes the hydrolysis of cADPR. These results suggest that NAD+ hydrolysis to ADPR and nicotinamide catalyzed by the NAD+ glycohydrolase occurs through the formation of a cADPR. E. cADP-ribosyl complex. The low production of cADPR by NAD+ glycohydrolase compared with invertebrate ADP-ribosyl cyclase is believed to be attributable to the fast hydrolysis of cADPR by the allosteric effect of cADPR bound to the same enzyme that produces it.

Adenosine Diphosphate Ribose↗

PubMed1998

Modulation of apoptotic cell death by extracellular matrix proteins and a fibronectin-derived antiadhesive peptide.

Cell adhesion to the extracellular matrix (ECM) has been implicated in apoptosis in anchorage-dependent cell types. We recently found that a peptide derived from fibronectin (termed III14-2) inhibits the integrin-mediated cell adhesion to ECM. Using this antiadhesive peptide and a variety of ECM proteins, we show here a critical role of the integrin-ECM protein interaction in apoptotic regulation of human umbilical vein endothelial cells (HUVEC). HUVEC in suspension underwent apoptosis under the serum-free conditions, as judged by nuclear and DNA fragmentations. This apoptosis was suppressed to varying degrees when alpha 5 beta 1, alpha v beta 3, and alpha 2 beta 1 integrins were occupied with either soluble or immobilized ECM proteins such as fibronectin, vitronectin, and type I collagen, respectively. Peptide III14-2, which had no effect by itself on the HUVEC apoptosis, disrupted the ligation of alpha 5 beta 1 and alpha v beta 3 but no alpha 2 beta 1 and ultimately led the cells to apoptosis, indicating that this antiadhesive peptide indirectly induces apoptosis by blocking cell survival signal delivered from alpha 5 beta 1 and alpha v beta 3 integrins. Genistein, a protein tyrosine kinase inhibitor, slightly reduced the rescuing effect of fibronectin, whereas sodium orthovanadate and bombesin, which increase in the level of protein tyrosine phosphorylation, made HUVEC less susceptible to apoptosis and blocked the effect of peptide III14-2. HUVEC adhesion to fibronectin substrate raised the tyrosine phosphorylation level of focal adhesion kinase and the expression of cytoprotective Bcl-2 protein, both of which were reversed by the antiadhesive effect of peptide III14-2. Thus, the opposing effects of ECM proteins, including fibronectin and vitronectin, and peptide III14-2 on HUVEC apoptosis appear to be due to the opposing effects of these factors on the signaling pathway which includes tyrosine phosphorylation of FAK and Bcl-2 expression.

Amino Acid Sequence↗

PubMed1998

Cloning of cDNAs encoding porcine and human DNase II.

We report the molecular cloning of cDNAs encoding porcine and human DNase II and the genomic structure of the human DNase II gene. The full length cDNAs for porcine and human DNase II were isolated by polymerase chain reaction on the basis of amino acid sequences determined for the tryptic peptides of porcine liver DNase II. The porcine and human cDNAs contain 1095 and 1083 bp open reading frames, respectively, and encode 364 and 360 amino acid proteins with calculated molecular masses of 40,157 and 39,555, respectively. The amino acid sequencing of purified porcine DNase II reveals two N-termini with corresponding sequences present within the same open reading frame, suggesting proteolytic processing for the covalently bonded subunit structure of DNase II. Northern blot analysis demonstrated that a single transcript of 2.0 kb mRNA coding for DNase II is ubiquitously expressed in human tissues. A database search revealed that the human genomic sequence of chromosome 19p13.2 contains the DNase II gene. Characterization of the genomic sequence showed that the DNase II gene consists of six exons separated by five introns whose splice acceptor/donor sites agree with the GT/AG rule.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase gamma.

An endonuclease named DNase gamma has been purified from the nuclei of apoptotic rat thymocytes [Shiokawa, Ohyama, Yamada and Tanuma (1997) Biochem. J. 326, 675-681]. Here we report the molecular cloning of a cDNA encoding a 35 kDa precursor protein for rat DNase gamma. A 1.6 kb mRNA coding for the DNase gamma precursor is detected at high levels in spleen, lymph nodes, thymus and liver. By using reverse transcriptase-mediated PCR, expression of DNase gamma mRNA is observed in kidney and testis but not in brain or heart. Analysis of recombinant DNase gamma reveals that full-length DNase gamma, including the N-terminal precursor, is an inactive proenzyme. The mature form of recombinant DNase gamma, from which the N-terminal precursor has been removed, has the same properties as purified DNase gamma: requirement for divalent cations, dependence on pH, sensitivity to Zn2+, and cleavage of chromosome DNA to nucleosomal units. In HeLa S3 cells stably transfected with the DNase gamma cDNA, exogenously introduced DNase gamma is activated by apoptotic stimuli; enhancement of DNA fragmentation, chromatin condensation and nuclear collapse are observed. These findings provide evidence for the involvement of DNase gamma in DNA fragmentation and nuclear structural changes during apoptosis.

Animals↗

PubMed1998

Identification and characterization of a tannic acid-responsive negative regulatory element in the mouse mammary tumor virus promoter.

Tannic acid, which comprises polyphenolic compounds from tea leaves, suppresses the glucocorticoid-induced gene expression of mouse mammary tumor virus (MMTV) integrated into 34I cells. To investigate whether this suppression is due to promoter responsiveness to tannic acid, we performed chloramphenicol acetyltransferase analysis transfecting a MMTV promoter containing a chloramphenicol acetyltransferase expression vector into mouse fibroblast L929 cells. Deletion analysis of the promoter region revealed that a 50-base pair (bp) region located downstream of the TATA element is responsible for the suppressive effect of tannic acid. The tannic acid-sensitive suppressibility was introduced into a thymidine kinase promoter by inserting the 50-bp region into the region on the 5'-upstream side of the promoter. Detailed point mutation analyses revealed that two elements, a 13-bp element and an ACTG motif in the 50-bp region, contribute to tannic acid sensitivity and promoter repressibility, respectively. Interestingly, this repressive ACTG motif is found in the human immunodeficiency virus promoter, the activity of which is also suppressed by tannic acid (Uchiumi, F., Maruta, H., Inoue, J., Yamamoto, T., and Tanuma, S. (1996) Biochem. Biophys. Res. Commun. 220, 411-417). Furthermore, electrophoretic mobility shift analysis revealed that a protein factor(s) in nuclear extracts from L929 cells binds to the 50-bp region in a sequence-specific manner and that the amount of DNA-protein complex is increased by tannic acid treatment. Moreover, the negative regulatory sequence ACTG and the tannic acid-sensitive 13-bp element in this region were shown to be responsible for the formation of the DNA-protein complex by electrophoretic mobility shift analysis and footprint analyses. These findings suggest that the suppressive effect of tannic acid on MMTV gene expression is mediated by a protein factor(s) that binds to the negative regulatory element containing the common ACTG motif in a cooperative manner with the tannic acid-sensitive 13-bp element.

Animals↗

PubMed1998

Mesangial cell apoptosis induced by a fibronectin fragment.

We previously showed that in passive Heymann nephritis (PHN) rats, a large quantity of fibronectin (FN) fragments containing the central cell-binding (CCB) domain and adjacent domains are generated in the kidney and excreted into urine (Nishizawa et al., Biol Pharm Bull 1998; 21: 429-433). To ascertain whether the FN fragments could affect the progression of PHN, we investigated the effect of a 150 K FN fragment containing the CCB and carboxyl-terminal heparin-binding (Hep 2) domains on cultured rat mesangial cells. When rat mesangial cells cultured on FN-coated plates were exposed to the 150 K FN fragment, some mesangial cells detached from the FN substrate and then underwent apoptosis as judged by nuclear and DNA fragmentations. The 150 K FN fragment competitively inhibited the mesangial cell adhesion to the FN substrate in a dose-dependent manner. Furthermore, gelatinzymography of the conditioned medium of mesangial cells showed that the 150 K FN fragment induced and/or potentiated the extracellular matrix (ECM)-degrading proteinases including matrix metalloproteinases (MMPs) of mesangial cells. These results indicate that the 150 K FN fragment may elicit mesangial cell apoptosis by disrupting the mesangial cell adhesion through two distinct ways: the inhibition of mesangial cell adhesion and the ECM-degradation by the 150 K FN fragment-induced MMPs. Thus, FN fragments containing the CCB and adjacent domains generated in the kidneys of PHN rats may be involved in the evolution of the renal injury.

Journal Article↗

PubMed1998

Presence of DNase gamma-like endonuclease in nuclei of neuronal differentiated PC12 cells.

DNase gamma, which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase gamma-like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca(2+) and Mg(2+), and is sensitive to Zn(2+). The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase gamma from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase gamma-like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.

Journal Article↗

PubMed1998

Purification and characterization of a DNase gamma-like endonuclease from Xenopus laevis liver.

We recently found that two apoptotic DNase gamma-like endonucleases (36 and 38 kDa DNases) were present in Xenopus laevis larval and adult liver cell nuclei and that their activities increased in metamorphic climax. Here, we purified the main DNase gamma-like endonuclease from Xenopus laevis liver cell nuclei and characterized its physical and enzymatic properties in detail. The molecular mass of Xenopus liver nuclear endonuclease was 38,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 35,000 was estimated by gel filtration. The purified Xenopus liver endonuclease was a neutral one and required both Ca2+ and Mg2+ for DNase activity. Unlike the mammalian DNase gamma, the Ca2+/Mg2+ requirement could not be supplied by Mn2+. The inhibition profiles by aurintricarboxylic acid, sodium citrate and divalent metal ions such as Co2+, Ni2+, Cu2+ and Zn2+ were similar to those of mammalian DNase gamma. These results suggest that this endonuclease is a Xenopus laevis homolog of the mammalian apoptotic endonuclease DNase gamma.

Journal Article↗

PubMed1998

cDNA cloning of human DNase gamma: chromosomal localization of its gene and enzymatic properties of recombinant protein.

We report here on the nucleotide sequence of the cDNA encoding human DNase gamma, which is a candidate for an apoptotic endonuclease. The cDNA clone isolated from a human spleen cDNA library is composed of a 918 bp open reading frame encoding a 305 amino acid precursor protein for DNase gamma. Northern blot analysis reveals that the expression of a single transcript of 1.5 kb DNase gamma mRNA is detected in the spleen and liver. The chromosomal localization of DNase gamma gene is mapped to chromosome 3 at region p21.1-p14.2 by fluorescence in situ hybridization (FISH). Characterization of thioredoxin-DNase gamma fusion protein (Trx-hDNase gamma) shows that the recombinant protein has a Ca(2+)/Mg(2+)- or Mn(2+)-dependent endonuclease activity that cleaves chromatin DNA to nucleosomal units. The optimum pH is around 7.2. Zn(2+) and aurintricarboxylic acid (ATA) inhibits the activity in dose-dependent manners. These properties are identical to those of purified DNase gamma.

Journal Article↗

PubMed1998

[Mammalian proteins that associate with telomeres].

Telomeres are the DNA-protein complexes found at the ends of linear chromosomes. The structure is believed to be important for chromosome stability and cell integrity, and thereby for cell senescence and immortality. Telomeric DNA consists of tandemly repeated sequences which are observed from human to yeast. For example, human and Saccharomyces telomeres have T2AG3 and TG1-3 repeats, respectively. Recently, various protein factors including replication factor C (RFC) have been found as telomere repeat sequence binding-proteins. Characterization of these proteins and their interactions with telomeres may provide a new insight into not only telomere functions but also aging, immortality and apoptosis of the cells.

DNA-Binding Proteins↗

PubMed1997

Purification, characterization, and amino acid sequencing of DNase gamma from rat spleen.

An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized. We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides. The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 30,000 was estimated by gel filtration. Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ alone and inhibited by Co2+, Ni2+, Cu2+, and especially Zn2+. Maximal activity was achieved at pH 7.2 in Mops-NaOH buffer. The sequence data on the NH2-terminal and seven internal peptides obtained by sequential digestions with Achromobacter protease I and endoproteinase Asp-N revealed that DNase gamma is a novel endonuclease that shows sequence homology with DNase I.

Amino Acid Sequence↗

PubMed1997

Purification and properties of DNase gamma from apoptotic rat thymocytes.

We previously identified three distinct DNA endonucleases, DNases alpha, beta and gamma, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, gamma-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase gamma to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase gamma exhibited one predominant protein band on SDS/PAGE and an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase gamma was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine and lysine residues. The N-terminal amino acid sequence of DNase gamma was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase gamma requires both Ca2+ and Mg2+ for activity. This requirement could be partially supplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ and Zn2+ inhibited DNase gamma activity. These bivalent cations also suppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X-rays. The same order of inhibitory ability was observed for these bivalent metal ions in vivo (in intact cells) and in vitro, suggesting that the suppression of apoptotic DNA fragmentation at the cellular level is due to the inhibition of DNase gamma. DNase gamma activity was found to exist at high levels in spleen, lymph node, thymus, liver and kidney, but little was present in brain, heart or pancreas. On the basis of these findings, together with previous data, we conclude that DNase gamma is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.

Animals↗

PubMed1997

Inhibitors of poly(ADP-ribose) polymerase suppress nuclear fragmentation and apoptotic-body formation during apoptosis in HL-60 cells.

The effects of 3-aminobenzamide (3ABm) and benzamide (BAm), known specific inhibitors of poly(ADP-ribose) polymerase (PARP), on actinomycin D (Act D)-induced apoptosis in HL-60 cells were examined. These inhibitors had no appreciable effect on apoptotic DNA fragmentation, chromatin condensation or PARP restriction cleavage, but clearly inhibited morphological changes, especially nuclear fragmentation and apoptotic-body formation, in a dose-dependent manner. These results suggest that the synthesis of ADP-ribose polymers is not essential for the progression of apoptotic DNA fragmentation and chromatin condensation, but is required in the processes leading to nuclear fragmentation and the subsequent apoptotic-body formation during apoptosis in HL-60 cells.

Aminobenzoates↗

PubMed1997

Role of (ADP-ribose)n catabolism in DNA repair.

Poly(ADP-ribose) is a reversible covalent-modifier of chromosomal proteins in eukaryotic cells. The function of poly(ADP-ribose) is not clear, although it has been suggested to be involved in the regulation of DNA transactions such as replication, repair, and transcription. Here we describe a specific competitive inhibitor of poly(ADP-ribose) glycohydrolase, a macrocircular ellagitannin oenothein B, and a nuclear system prepared from synchronized HeLa S3 cells at mid-G1 phase that enable us to examine the role of poly(ADP-ribose) catabolism in DNA repair. The results suggest that poly(ADP-ribose) is capable of generating ATP by the concerted action of poly(ADP-ribose) glycohydrolase and ADP-ribose pyrophosphorylase and that this ATP enables repair DNA synthesis.

Adenosine Triphosphate↗

PubMed1997

Occurrence of DNase gamma-like apoptotic endonucleases in hematopoietic cells in Xenopus laevis and their relation to metamorphosis.

DNase gamma has been suggested to be an endonuclease responsible for thymic apoptosis in mammals. Using the frog, Xenopus laevis, we examined whether any hematopoietic cells other than thymocytes have DNase gamma activity. The activity gel assays and HeLa cell nuclear assays for DNase revealed that nuclei from red blood cells and cells in liver (an erythropoietic organ) contain DNase gamma-like activities (molecular mass of 38 and 36 kDa) indicated by their biochemical features (internucleosomal DNA cleavage, requirement of Ca2+ and Mg2+, and sensitivity to Zn2+). Distribution of these two (38 and 36 kDa) forms of enzyme was determined by cell fractionation analysis of liver cells: the 38 kDa form of enzyme was found in erythrocytes/erythroblasts and hepatocytes, whereas the 36 kDa form was in lymphocytes/macrophages. Interestingly, these two enzyme activities increased during metamorphic climax, at which time larval-type cells are converted to adult ones. It thus appears that these gamma-type DNases are involved in programmed cell death during metamorphosis.

Animals↗

PubMed1997

The restrictive proteolysis of alpha-fodrin to a 120 kDa fragment is not catalyzed by calpains during thymic apoptosis.

The alpha-subunit (240 kDa) of fodrin was found to be digested selectively to a 120 kDa fragment during apoptosis of rat thymocytes in vivo and in vitro. This fragment was detected by an antibody (Ab) against full length alpha-fodrin, but not by the anti-N-terminal sequence (GMMPR) of the mu-calpain-generated 150 kDa fragment Ab or the anti-PEST sequence of alpha-fodrin Ab. On the other hand, basal levels of the 150 kDa fragment were constantly recognized by these three antibodies during apoptosis. The production of the 120 kDa fragment during apoptosis was not affected by the addition of calpain inhibitors such as Ac-LLLnal and E-64d, despite inhibition of the generation of the 150 kDa fragment. When x-irradiated thymocytes were incubated in the presence of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), both production of the 120 kDa fragment and apoptosis were suppressed. Purified mu- and m-calpain did not catalyze the formation of the 120 kDa fragment from purified alpha-fodrin in vitro. These results suggest that a protease different from calpains is involved in the major process of alpha-fodrin proteolysis to a 120 kDa fragment during thymic apoptosis.

Journal Article↗

PubMed1996

Replication factor C recognizes 5'-phosphate ends of telomeres.

Telomere structure is suggested to be important for chromosome and cell integrity and thereby for cell senescence and immortality. In a search for cDNA encoding proteins that bind specifically to telomere repeat sequences, we used random primer-labeled telomere probes to screen a lambda gt11 Jurkat cDNA library. The clone obtained encodes the central region of the large subunit of replication factor C (RFC), a known activator of DNA polymerase delta. Electrophoretic mobility shift analyses of the binding ability of RFC-glutathione S-transferase (GST) fusion protein to telomere probes revealed that RFC recognizes preferentially 5'-phosphoryl (P) groups but not 3'-hydroxyl (OH) groups at the ends of double-stranded telomere repeats. This structure-specific binding of RFC is supported by the observations that it binds to 3'-OH/5'-P ends in telomere repeats produced by DNase gamma, but not to those produced by 3'-P/5'-OH ends for DNase alpha. These findings suggest a novel function for RFC in telomere stability or turnover.

Binding Sites↗

PubMed1996

Properties of poly(ADP-ribose) glycohydrolase purified from pig testis nuclei.

A poly(ADP-ribose) glycohydrolase was purified more than 5,000-fold to apparent homogeneity from pig testis nuclei with a yield of 16%. A protein band, whose molecular mass (Mr) was estimated to be 58,000, detected by SDS-polyacrylamide gel electrophoresis of the purified preparation, was shown to have glycohydrolase activity upon assay by the renaturation method. A native Mr of 51,000 was determined by gel permeation. This polypeptide is a basic protein with a pI value of 8.8. The mode of hydrolysis of poly(ADP-ribose) [(ADP-ribose)n] by this enzyme is exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) is 5.4 microM and the Vmax of its hydrolysis is 34.5 micromol x min(-1) x mg protein(-1). The optimum pH for enzyme activity is 7.2. Low concentrations (50 approximately 150 mM) of monovalent salts stimulate the enzyme activity. The poly(ADP-ribose) glycohydrolase present in pig testis nuclei has some properties different from either nuclear poly(ADP-ribose) glycohydrolase (type I) or cytoplasmic poly(ADP-ribose) glycohydrolase (type II), purified previously from several tissues including pig thymus, guinea pig liver, calf thymus, human erythrocytes, and placenta. These differences suggest the tissue specificity of poly(ADP-ribose) glycohydrolase.

Animals↗