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Sei-ichi Tanuma

Publications and source records attributed to Sei-ichi Tanuma.

At least 19 recordsLinked to original sources
PubMed2006

A new fluorescent probe for zinc(II): an 8-hydroxy-5-N,N-dimethylaminosulfonylquinoline-pendant 1,4,7,10-tetraazacyclododecane.

A new fluorescent probe for Zn2+, namely, 8-hydroxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant cyclen (L8), was designed and synthesized (cyclen=1,4,7,10-tetraazacyclododecane). By potentiometric pH, 1H NMR, and UV spectroscopic titrations, the deprotonation constants pKa1-pKa6 of L(8)4 HCl were determined to be <2, <2, <2 (for amino groups of the cyclen and quinoline moieties), 7.19+/-0.05 (for 8-OH of the quinoline moiety), 10.10+/-0.05, and 11.49+/-0.05, respectively, at 25 degrees C with I=0.1 (NaNO3). The results of 1H NMR, potentiometric pH, and UV titrations, as well as single-crystal X-ray diffraction analysis, showed that L8 and Zn2+ form a 1:1 complex [Zn(H-1L8)], in which the 8-OH group of the quinoline ring of L8 is deprotonated and coordinates to Zn2+, in aqueous solution at neutral pH. On addition of one equivalent of Zn2+ and Cd2+, the fluorescence emission of L8 (5 microM) at 512 nm in aqueous solution at pH 7.4 [10 mM HEPES with I=0.1 (NaNO3)] and 25 degrees C increased by factors of 17 and 43, respectively. We found that the cyclen moiety has the unique property of quenching the fluorescence emission of the quinolinol moiety when not complexed with metal cations, but enhancing emission when complexed with Zn2+ or Cd2+. In addition, the Zn2+-L8 complex [Zn(H-1L8)] is much more thermodynamically and kinetically stable (Kd{Zn(H-1L8)}=[Zn2+]free[L8]free/[Zn(H-1L8)]=8 fM at pH 7.4) than the Zn2+ complexes of our previous Zn2+ fluorophores ([Zn(H-1L2)] and [Zn(L3)]). Furthermore, formation of [Zn(H-1L8)] is much faster than those of [Zn(H-1L2)] and [Zn(L3)]. The staining of early-stage apoptotic cells with L8 is also described.

Amines↗
PubMed2006

Action of apoptotic endonuclease DNase gamma on naked DNA and chromatin substrates.

The internucleosomal cleavage of genomic DNA is a biochemical hallmark of apoptosis. DNase gamma, a Mg2+/Ca2+-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases, but its biochemical characteristic has not been fully elucidated. Here, using recombinant DNase gamma, we showed that DNase gamma is a Mg2+/Ca2+-dependent single-stranded DNA nickase and has a high activity at low ionic strength. Under higher ionic strength, such as physiological buffer conditions, the endonuclease activity of DNase gamma is restricted, but its activity is enhanced in the presence of linker histone H1, which explains DNA cleavage at linker regions of apoptotic nuclei.

Apoptosis↗
PubMed2006

Structure basis for the inhibitory mechanism of a novel DNase gamma-specific inhibitor, DR396.

DNase gamma, a member of the DNase I family, has been suggested to cause DNA fragmentation during apoptosis. We recently identified 4-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel specific inhibitor for human DNase gamma [Sunaga, S.; Kobayashi, T.; Yoshimori, A.; Shiokawa, D.; Tanuma, S. Biochem. Biophys. Res. Commun.2004, 325, 1292]. However, the binding mode (coordinate) of DR396 to DNase gamma has not yet been defined. Here, we examined the molecular basis for the inhibitory activity of DR396 to DNase gamma by structure-based computational docking studies. In the blind-docking study using a human DNase gamma homology model, a unique binding site of DR396 was predicted, which is tentatively named the 'DNA trapping site' because of the binding domain of the unhydrolyzed DNA strand, but not the active site. Targeting the DNA trapping site as a hot spot, new human DNase gamma inhibitors were obtained from our diverse chemical library in silico. These inhibitors showed high correlations between their predicted binding-free energies (DeltaGs) and observed IC50 values in the DNA trapping site but not the active site. The IC50 of a regioisomer of DR396, 5-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DF365), was 73 microM (DeltaG=-9.75 kcal/mol), a 20-fold weaker inhibitory ability than that of DR396 (IC50=3.2 microM, DeltaG=-11.22 kcal/mol). Fluorescein and triazine derivatives, partial structures of DR396, had little inhibitory activity for DNase gamma. Docking analyses of the interaction between DR396 and DNase gamma revealed that DR396 binds tightly to three subsites (S1, S2, and S3) in the trapping site of DNase gamma by forming six hydrogen bonds, whereas DF365 and the partial structures are unable to form hydrogen bonds at all three subsites. These findings suggest that the specificity and potency of the inhibitory activity of DR396 for DNase gamma is due to the specific interaction of DR396 with three subsites in the DNA trapping site of DNase gamma.

Amino Acid Sequence↗
PubMed2005

Physical and biochemical properties of mammalian DNase X proteins: non-AUG translation initiation of porcine and bovine mRNAs for DNase X.

DNase X is the first human DNase protein identified as being homologous with DNase I. In the present study we describe the isolation of several mammalian DNase X cDNAs and the molecular characterization of their coding proteins. A sequence comparison reveals some conserved characteristics: all the mammalian DNase X proteins have an N-terminal signal peptide, a potential N-linked glycosylation site and a C-terminal hydrophobic domain. Human DNase X, ectopically expressed in HeLa S3 cells, is located in the ER (endoplasmic reticulum) and is modified by an N-linked glycosylation at Asn-243. Gene expression analyses show that the high expression level in muscular tissues, a known feature of human DNASE X, is also observed in mouse DNase X. Interestingly, the translation of porcine and bovine DNase X proteins occurs in the absence of an in-frame AUG initiation codon. We show that their mRNAs utilize a conserved CUG triplet for translation initiation.

Amino Acid Sequence↗
PubMed2005

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.

Amyloid Precursor Protein Secretases↗
PubMed2005

Involvement of DNase gamma in the resected double-strand DNA breaks in immunoglobulin genes.

Somatic hypermutation (SHM) of immunoglobulin variable (V) region genes occurs in the germinal center (GC) B cells during immune responses, depending on activation-induced cytidine deaminase (AID). SHM is associated with resected double-strand DNA breaks (DSBs) which were shown to occur specifically in rearranged V regions in the GC B cells and CD40-stimulated B cells expressing AID. So far, endonucleases responsible for the DSBs have not been identified. Here we show that DNase gamma, a member of DNase I family of endonucleases, is expressed in GC B cells and CD40-stimulated B cells. Overexpression of DNase gamma in the mutation-competent Ramos B-cell line resulted in a marked increase in the resected but not blunt DSBs in the V region. Conversely, a selective DNase gamma inhibitor, DR396, suppressed the generation of the resected DSBs. These results suggest that DNase gamma is involved in the generation of resected DSBs associated with SHM.

Animals↗
PubMed2004

Characterization of Smubp-2 as a mouse mammary tumor virus promoter-binding protein.

A cDNA encoding a rat Smubp-2 has been cloned from a lambdagt11 library by South-Western blot screening using a 50-bp tannic acid responsive element [J. Biol. Chem. 273 (1998) 12499] of the mouse mammary tumor virus (MMTV) promoter region as a probe. The full-length cDNA encodes a protein with a predicted size of 108 kDa. Northern blot analysis revealed that the gene expression of Smubp-2 is comparatively high in testis, moderate in brain, and low in other tissues. The recombinant Smubp-2 protein was expressed as a GST- or Trx-fusion protein in Escherichia coli and purified by affinity column chromatography. Gel mobility shift competition analysis indicated that the recombinant Smubp-2 protein binds to region II (containing the ACTG-motif) in the 50-bp element in the MMTV promoter. A transient transfection assay of the Smubp-2 expression vector with MMTV promoter-containing Luciferase (Luc) reporter plasmids into mouse cells suggested that Smubp-2 is a negative transcription factor. Furthermore, the MMTV promoter activity was suppressed in cells expressing high levels of Smubp-2. Insertion of the 50-bp element upstream of the SV40 promoter negatively responded to the induced expression of Smubp-2. These results suggest that the negative transcriptional effect of Smubp-2 arises from its binding to the 50-bp element located in the MMTV promoter region.

Amino Acid Sequence↗
PubMed2004

Expression of DNase gamma during Fas-independent apoptotic DNA fragmentation in rodent hepatocytes.

Endonuclease-induced DNA fragmentation is a hallmark of apoptosis. DNase gamma (DNase gamma) was recently identified as one of the endonucleases responsible for apoptotic DNA fragmentation. In this study, immunohistochemistry for DNase gamma was performed on paraffin sections of rodent liver in well-defined models of hepatocyte apoptosis induced by Fas antibody (Fas) or cycloheximide (CHX), and necrosis induced by lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). DNase gamma immunoreactivity was compared with TdT-mediated dUTP nick-end labeling (TUNEL) reactivity. Our results showed TUNEL reactivity in both apoptotic and necrotic hepatocytes. DNase gamma immunoreactivity was not detected during LPS-induced or CCl4-induced hepatocyte necrosis. In contrast, it was evident during CHX-induced, but not Fas-induced, apoptotic DNA fragmentation. These findings suggest that DNase gamma plays an important role in Fas-independent apoptotic DNA fragmentation in hepatocytes.

Animals↗
PubMed2004

Monitoring apoptosis with fluorescent Zn2+-indicators.

Apoptosis, a mechanism of programmed cell death that removes superfluous and harmful cells, is important both during development and in tissue homeostasis. Although Zn2+ is believed to be critical in apoptosis, the precise details of its role have yet to be elucidated. The macrocyclic Zn2+ ligand dansylamidoethylcyclen [L1*(HCl)4*(H2O)2], which is found primarily in a diprotonated form (H2L1), is cell-permeable and forms a strongly fluorescent 1:1 Zn2+ complex when Zn2+ entry into cells is facilitated by the Zn2+ ionophore pyrithione. H2L1 can be used to readily identify HeLa cells undergoing the early stages of etoposide-induced apoptosis because of the increased level of free Zn2+ that occurs at this time. The selectivity of H2L1 for the detection of apoptotic cells was verified by a conventional probe for apoptosis, annexin V-Cy3. Here, we describe methods for detecting apoptotic cells with H2L1 and for comparing detection of apoptosis with H2L1 to detection with annexin V-Cy3 and Zinquin.

Annexin A5↗
PubMed2004

Changes in the activities and gene expressions of poly(ADP-ribose) glycohydrolases during the differentiation of human promyelocytic leukemia cell line HL-60.

The metabolism of poly(ADP-ribose) is known to play important roles in the nuclear function of the mammalian cells. In this study, changes in the activities and gene expressions of poly(ADP-ribose) glycohydrolases (PARG) in HL-60 cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or a PARG inhibitor, tannic acid, were investigated. Nuclear PARG activities of HL-60 cells treated with TPA were reduced to 30-40% of the activity in untreated cells at 24 h, while PARG activities in the cytoplasm remained unchanged. The transient decrease in the nuclear PARG activity by TPA treatment was accompanied by differentiation as measured by the nitroblue tetrazolium (NBT) reducing activity and adhesion to the culture dishes. In the presence of H7, an inhibitor of protein kinase C (PKC), both the decrease in nuclear PARG activity and the induction of differentiation by TPA treatment were suppressed. On the other hand, treatment with tannic acid caused the nuclear PARG activity to decrease continuously while the NBT reducing activity increased, but no morphological differentiation to macrophage-like cells was apparent. In order to analyze PARG gene expression, we isolated the human PARG cDNA by the RT-PCR technique. RT-PCR analysis revealed that TPA treatment leads to a reduction in the PARG gene expression prior to the phenotypic expression of macrophage-like cell differentiation, which was diminished by the presence of H7. Also, PARG gene expression was reduced by tannic acid treatment. These results provide the first evidence that a transient decrease in nuclear PARG activity is important for the onset of differentiation of HL-60 cells to macrophage-like cells.

Cell Adhesion↗
PubMed2004

Genomic drug discovery for apoptosis regulation using a new computer screening amino acid complement wave method.

A novel caspase-3-specific inhibitory peptide and an agonistic peptide that binds to the Fas molecule were discovered using our computer screening strategy called the amino acid complement wave (ACW) method, which is based on the complementarities of interacting amino acids between comprehensive testing peptides and a target protein surface pocket. The precise binding configurations of the designed peptides on the three-dimensional (3D) structure of the target protein and the prediction of binding affinities (DeltaG) are determined by the molecular docking program. A designed novel tetrapeptide inhibitor of caspase-3, Ac-DNLD-CHO, was revealed to have potent and specific inhibitory activity. When a designed Fas ligand mimic peptide (Fas reactive peptide-4, FRAP-4) was multimerized by carboxyl terminal linkages of polylysine branches (MAP), the octamer (FRAP-4)(8)-MAP effectively induced apoptosis of human ovarian cancer cell line NOS4 cells. Thus the ACW method for structure-based design of optimized small peptides can be used to further develop small peptidomimetic and nonpeptidic organic forms into a new generation of effective pharmaceuticals.

Amino Acid Sequence↗
PubMed2003

Identification of two functional nuclear localization signals in DNase gamma and their roles in its apoptotic DNase activity.

Among DNase I family members, only DNase gamma causes DNA fragmentation during apoptosis. However, the molecular basis for this functional feature of DNase gamma is poorly understood. Here we describe the identification of functional NLSs (nuclear localization signals) in DNase gamma and their roles in its apoptotic function. DNase gamma contains two NLSs: a classical bipartite-type NLS (NLS1) located in the N-terminal half, and a short basic domain (NLS2) at the C-terminus. No potential NLSs are found in the primary structures of other DNase I family DNases. Inactivation of either NLS1 or NLS2 causes reduced DNA ladder-producing activity in DNase gamma. Disruption of NLS2 suppresses ladder formation more effectively than disruption of NLS1. DNase gamma doubly mutated in both NLSs is enzymically active, but no longer catalyses apoptotic DNA fragmentation. Although DNase I fails to produce ladder formation during apoptosis, DNase I fused to NLS2 of DNase gamma through its C-terminus is able to catalyse DNA fragmentation in apoptotic cells. These results indicate that the presence of either NLS1 or NLS2 is necessary for the apoptotic function of DNase gamma, and that the most important domain for this function is NLS2. These findings also explain the lack of apoptotic DNase activity in the other DNase I family DNases.

Animals↗
PubMed2003

Subcellular localization of poly(ADP-ribose) glycohydrolase in mammalian cells.

Posttranslational modification plays important roles in a range of cellular functions. Poly(ADP-ribosyl)ation influences DNA repair, transcription, centrosome duplication, and chromosome stability. Poly(ADP-ribose) attached to acceptor proteins should be properly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). However the subcellular localization and the role of PARG have not been well characterized. Here, we transiently expressed GFP- or Myc-tagged human PARG in mammalian cells and revealed that the subcellular distribution of human PARG changes dramatically during the cell cycle. GFP-hPARG is found almost exclusively in the nucleus during interphase. During mitosis, most GFP-hPARG protein localizes to the cytoplasm and hardly any GFP-hPARG protein is found associated with the chromosomes. Furthermore, we found that GFP-hPARG localizes to the centrosomes during mitosis. Our findings suggest that shuttling of PARG between nucleus and cytoplasm and proper control of poly(ADP-ribose) metabolism throughout the cell cycle may play an important role in regulating cell cycle progression and centrosome duplication.

3T3 Cells↗
PubMed2003

Putative function of ADAM9, ADAM10, and ADAM17 as APP alpha-secretase.

The putative alpha-secretase cleaves the amyloid precursor protein (APP) of Alzheimer's disease in the middle of the amyloid beta peptide (Abeta) domain. It is generally thought that the alpha-secretase pathway mitigates Abeta formation in the normal brain. Several studies have suggested that ADAM9, ADAM10, and ADAM17 are candidate alpha-secretases belonging to the ADAM (a disintegrin and metalloprotease) family, which are membrane-anchored cell surface proteins. In this comparative study of ADAM9, ADAM10, and ADAM17, we examined the physiological role of ADAMs by expressing these ADAMs in COS-7 cells, and both "constitutive" and "regulated" alpha-secretase activities of these ADAMs were determined. We tried to suppress the expression of these ADAMs in human glioblastoma A172 cells, which contain large amounts of endogenous alpha-secretase, by lipofection of the double-stranded RNA (dsRNA) encoding each of these ADAMs. The results indicate that ADAM9, ADAM10, and ADAM17 catalyze alpha-secretory cleavage and therefore act as alpha-secretases in A172 cells. This is the first report that to suggest the endogenous alpha-secretase is composed of several ADAM enzymes.

ADAM Proteins↗
PubMed2003

Transcriptional suppression of the HIV promoter by natural compounds.

Tannins and lignins are natural compounds contained in plants such as tea leaves. Previously, we demonstrated that tannic acid represses 12-o-tetra-decanoyl phorbol-13-acetate (TPA)-induced human immunodeficiency virus (HIV) promoter activity. Furthermore, we demonstrated that a 30-bp element located just downstream of the NF-kappaB element in the HIV promoter responds negatively to tannic acid. However, the kinds of molecules responsible for this suppressive effect have remained unknown, because tannic acid is a mixture of various galloylglucoses. Here, we examined structure-defined natural compounds for HIV promoter-suppressive effects. We found that ellagitannins suppress TPA-induced HIV promoter activity to the same extent as tannic acid. 3-phenylcoumarins, isoflavone and chalcones have more suppressive effects than ellagitannins. On the other hand, other flavonoids and acetogenins have no suppressive effect. 3-phenylcoumarins and chalcones showed no suppressive effect on the cytomegalovirus (CMV) promoter, suggesting that they act specifically on the HIV promoter. These results suggest that 3-phenylcoumarin or chalcone compounds could be used to develop novel anti-HIV drugs with an action targeted at HIV promoter activity.

Anti-HIV Agents↗
PubMed2003

Apoptotic DNA endonuclease (DNase-gamma) gene transfer induces cell death accompanying DNA fragmentation in human glioma cells.

AIMS: Both the genetic restoration of the apoptotic pathway and the introduction of proapoptotic molecules are now drawing attention. Concerning apoptosis of human glioma cells induced by human interferon-beta protein, we found that DNA endonuclease (DNase-gamma) acts as an executive molecule. The authors investigated whether gene transfer of this DNase-gamma exerts some therapeutic effects on human glioma cells. METHODS: We transduced U251SP, U251MG, and T98G human glioma cells with DNase-gamma gene via multilamellar cationic liposomes, monitored the growth of those cells, and carefully observed the cell-death pattern. RESULTS: DNase-gamma gene transfer resulted in an overexpression of DNase-gamma protein and induced DNA fragmentation in gene-transferred cells. The cytotoxic effect rose with multiple inoculations of the liposome, suggesting a relationship between its expression and the therapeutic effect. CONCLUSIONS: These results demonstrate that DNase-gamma gene transfer can induce apoptosis in human glioma cells, indicating its potential to become a future gene therapy strategy.

Apoptosis↗