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Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 73 records · Page 4Linked to original sources
PubMed2000

Interlaboratory comparison of the CB6F1-Tg rasH2 rapid carcinogenicity testing model.

Several genetically engineered mouse models are currently being examined for potential use in cancer hazard identification. We have undertaken an interlaboratory comparison of the performance of the CB6F1-Tg rasH2 transgenic mouse in cancer bioassays concurrently conducted in the United States and Japan. Chemicals selected for study included known human carcinogens (melphalan and cyclosporin A) and known rodent carcinogens (p-cresidine and vinyl carbamate) tested at carcinogenic doses, and non-carcinogens (p-anisidine and resorcinol) tested at appropriate high doses. Because of abdominal adhesions caused by the intraperitoneal dosing vehicle, melphalan was excluded from the study results. The remaining five studies showed similar results between the two laboratories conducting each study. Vinyl carbamate gave the strongest positive response inducing lung adenomas and carcinomas and splenic hemangiosarcomas. p-Cresidine was considered positive for urinary bladder transitional neoplasia. Cyclosporin A, p-anisidine, and resorcinol were negative in all studies. Although only five chemicals were successfully tested in this interlaboratory comparison, there was good concordance in outcome for the strong carcinogens and for the non-carcinogens. Successful testing of chemicals with less carcinogenic potential may require modifications in study design to include more animals and longer study duration.

Adenoma↗
PubMed2000

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.

Aldehyde Dehydrogenase↗
PubMed2000

Requirement for canonical base pairing in the short pseudoknot structure of genomic hepatitis delta virus ribozyme.

The tertiary structure of the 3'-cleaved product of the genomic hepatitis delta virus (HDV) ribozyme was solved by X-ray crystallographic analysis. In this structure, three single-stranded regions (SSrA, -B and -C) interact intricately with one another via hydrogen bonds between nucleotide bases, phosphate oxygens and 2'-OHs to form a nested double pseudoknot structure. Among these interactions, two Watson-Crick (W-C) base pairs, 726G-710C and 727G-709C, that form between SSrA and SSrC (P1.1) seem to be especially important for compact folding. To characterize the importance of these base pairs, ribozymes were subjected to in vitro selection from a pool of RNA molecules randomly substituted at positions 709, 710, 726 and 727. The results establish the importance of the two W-C base pairs for activity, although some mutants are active with one G-C base pair. In addition, the kinetic parameters were analyzed in all 16 combinations with two canonical base pairs. Comparison of variant ribozymes with the wild-type ribozyme reveals that the difference in reaction rates for these variants (DeltaDelta G (double dagger)) is not simply accounted for by the differences in the stability of P1.1 (DeltaDelta G (0)(37)). The role played by Mg(2+)ions in formation of the P1.1 structure is also discussed.

Base Pairing↗
PubMed2000

OUT, a novel basic helix-loop-helix transcription factor with an Id-like inhibitory activity.

Transcription factors belonging to the basic helix-loop-helix (bHLH) family are involved in various cell differentiation processes. We report the isolation and functional characterization of a novel bHLH factor, termed OUT. OUT, structurally related to capsulin/epicardin/Pod-1 and ABF-1/musculin/MyoR, is expressed mainly in the adult mouse reproductive organs, such as the ovary, uterus, and testis, and is barely detectable in tissues of developing embryos. Physical association of OUT with the E protein was predicted from the primary structure of OUT and confirmed by co-immunoprecipitation. However, unlike other bHLH factors, this novel protein failed to bind E-box or N-box DNA sequences and inhibited DNA binding of homo- and heterodimers consisting of E12 and MyoD in gel mobility shift assays. In luciferase assays, OUT inhibited the induction of E-box-dependent transactivation by MyoD-E12 heterodimers. Deletion studies identified the domain responsible for the inhibitory action of OUT in its bHLH and C-terminal regions. Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of OUT. These inhibitory functions of OUT closely resemble those of the helix-loop-helix inhibitor Id proteins. Based on these findings, we propose that this novel protein functions as a negative regulator of bHLH factors through the formation of a functionally inactive heterodimeric complex.

Amino Acid Sequence↗
PubMed2000

A novel snail-related transcription factor Smuc regulates basic helix-loop-helix transcription factor activities via specific E-box motifs.

Snail family proteins are zinc finger transcriptional regulators first identified in Drosophila which play critical roles in cell fate determination. We identified a novel Snail -related gene from murine skeletalmusclecells designated Smuc. Northern blot analysis showed that Smuc was highly expressed in skeletal muscle and thymus. Smuc contains five putative DNA-binding zinc finger domains in its C-terminal half. In electrophoretic mobility shift assays, recombinant zinc finger domains of Smuc specifically bound to CAGGTG and CACCTG E-box motifs (CANNTG). Because basic helix-loop-helix transcription factors (bHLH) bind to the same E-box sequences, we examined whether Smuc competes with the myogenic bHLH factor MyoD for DNA binding. Smuc inhibited the binding of a MyoD-E12 complex to the CACCTG E-box sequence in a dose-dependent manner and suppressed the transcriptional activity of MyoD-E12. When heterologously targeted to the thymidine kinase promoter as fusion proteins with the GAL4 DNA-binding domain, the non-zinc finger domain of Smuc acted as a transcriptional repressor. Furthermore, overexpression of Smuc in myoblasts repressed transactivation of muscle differentiation marker Troponin T. Thus, Smuc might regulate bHLH transcription factors by zinc finger domains competing for E-box binding, and non-zinc finger repressor domains might also confer transcriptional repression to control differentiation processes.

Amino Acid Sequence↗
PubMed2000

Characterization of the selective staining of DNA on gels using two fluorochromes.

Ethidium bromide (EB) and 4'-6-diamidino-2-phenylindole (DAPI) are both well-known fluorochromes for detecting DNA fragments. EB binds to DNA by intercalation and DAPI binds in the DNA minor groove. We previously developed a staining method using both EB and DAPI that is selective for AT-rich DNA fragments. Using this double-staining method, AT-rich DNA fragments are visualized as bluish-white fluorescent bands. To further characterize this method, a series of synthetic DNA fragments were designed with systematic variations in the length, AT content, and DNA sequence pattern. The staining properties of these fragments were determined in the presence of DAPI and EB, and the following results were obtained. (i) In a series of fragments with three AT base pairs followed by one GC base pair, the stained DNA fragments exhibited different fluorescent colors and varied from bluish (more DAPI staining) to pinkish (less DAPI staining) in the order 5'-AAA-3', 5'-AAT-3', 5'-ATA-3', 5'-TTA-3'. (ii) In fragments with constant AT content, the blue fluorescent color increased with increasing number of A (or T) nucleotides, due to increased DAPI binding. The blue color was saturated when the number of A (or T) nucleotides was 12 or greater. (iii) The fluorescent color of the stained DNA fragments changed in the order of red-orange, pink, pinkish-white, white, bluish-white, blue as the AT content increased from 0 to 100%. Thus, the fluorescent color of DNA fragments stained with DAPI and EB depends on base composition and nucleotide sequence, suggesting that individual stained DNA fragments may have characteristic and specific fluorescent colors. The fluorescent color emitted by specific stained DNA fragments in the presence of EB and DAPI can be analyzed with a high degree of sensitivity and resolution using the XYZ colorimetric system.

Base Pairing↗
PubMed2000

A case of malignant lymphoma of the hand.

Malignant lymphoma is rarely seen in the hand. We present a case of malignant lymphoma of the hand with Ga-67 citrate and MR images, and discuss the usefulness of Ga-67 citrate scintigraphy for diagnosing this condition.

Aged↗
PubMed2000

Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.

We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.

Animals↗
PubMed2000

Synthesis and cytokinin activity of fluorescent 7-Phenylethynylimidazo[4,5-b]pyridine and its riboside.

7-Phenylethynylimidazo[4,5-b]pyridine and its riboside have been newly developed as fluorescent carbon-substituted cytokinin analogues. Palladium-catalyzed coupling of 7-iodo-3-(tri-O-acetyl-beta-D-ribofuranosyl)imidazo[4,5-b]pyridine with phenylacetylene followed by ammonolysis afforded the 7-phenylethynyl riboside via its tri-O-acetate. Acid hydrolysis of the riboside provided its free base, which showed a marked enhancement in fluorescence intensity in an aqueous alkaline solution. The free base and its riboside were more active than the corresponding 6-phenylethynylpurine and its riboside, respectively, in Amaranthus betacyanin and tobacco callus bioassays. Surprisingly, the imidazo[4,5-b]pyridine base exhibited strong cytokinin activity comparable to that of N(6)-benzyladenine in the tobacco callus bioassay. This compound would be useful for studying localization and transport of cytokinins in cells or tissues of plants.

Cytokinins↗
PubMed2000

Crystal structure and conformation of beta-substituted (Z)- and (E)-6-styrylpurines as conformer models.

Crystal structures of conformationally restricted (Z)- and (E)-6-styrylpurines with the beta-substituents involving hydrogen, chlorine, and bromine atoms as well as a methylthio group were studied as conformer models of N(6)-adenines in relation to active conformation of cytokinins. X-ray crystallographic analyses confirmed that all of the trans-isomers exist in an anti conformation, whereas the cis-isomers except the (E)-methylthio derivative adopt a syn conformation. The derivative with a bulky beta-substituent was found to be in an anti conformation in contrast to the other cis-isomers. The preferred anti conformation and potent cytokinin activity of the trans-isomers supports the anti-transoid form as the most plausible active conformation of N(6)-adenines. In addition, it is likely that the syn-cisoid form of N(6)-adenines is also involved in receptor binding, by considering both the preferred syn conformation of the cis-isomers and their moderate activity, although it does not play a major role compared to the anti-transoid form.

Crystallography, X-Ray↗
PubMed2000

A novel RNA motif that binds efficiently and specifically to the Ttat protein of HIV and inhibits the trans-activation by Tat of transcription in vitro and in vivo.

BACKGROUND: To find a novel RNA that would bind efficiently and specifically to Tat protein but not to other cellular factors, we used an in vitro selection method and isolated a novel aptamer RNATat, a 37-mer RNA oligomer, that binds efficiently to the Tat protein of HIV-1. In the present study, we analysed various properties of aptamer RNATat, including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function. RESULTS: The binding affinity of the isolated aptamer RNATat to Tat-1 was 133 times higher than that of authentic TAR-1 RNA. RNATat is composed of inverted repeats of two TAR-like motifs, and even though RNATat had two Tat-binding core elements, the interaction with Tat took place at a molar ratio of 1 : 1. Several functional groups of aptamer RNATat responsible for Tat binding were identified. The selected aptamer RNATat competed effectively for binding to Tat even in the presence of a large excess of TAR-1 or TAR-2 RNA in vitro, and specifically prevented Tat-dependent trans-activation both in vitro and in vivo. CONCLUSIONS: Our results indicate that a novel aptamer, RNATat, retained strong affinity for Tat even in the presence of a large excess of HIV TAR. RNATat binds efficiently to Tat proteins or peptides derived from either HIV-1 or HIV-2. Unlike TAR RNA, RNATat affinity does not depend upon cellular proteins such as cyclin T1, thus RNATat has the potential for use as a molecular recognition element in biosensors.

Base Sequence↗
PubMed2000

Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease.

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DeltaNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DeltaNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DeltaNS3 with a binding constant of about 10 nM and inhibit approximately 90% of the protease activity of DeltaNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DeltaNS3 with a Ki approximately 100 nM in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.

Base Sequence↗
PubMed2000

Analysis of interaction between RNA aptamer and protein using nucleotide analogs.

Non-structural protein 3 (NS3) derived from Hepatitis C virus (HCV) is essential for viral proliferation and has two functional domains; trypsin-like serine protease and helicase. Recently we obtained three types of RNA aptamers (G9-I, -II and -III) bound to NS3 protease domain (delta NS3) by in vitro selection and confirmed their strong inhibition for protease activity. These aptamers have a common sequence, 5'-GA(A/U)UGGGAC-3', forming a loop structure by Mulfold secondary structure modeling. G9-I shows a three-way junction and G9-II and -III have four-way junction structures. To characterize the active structure of these aptamers, we applied modification interference analysis using nucleotide analogs and identified common important nucleotides in these three aptamers.

Base Sequence↗
PubMed2000

Analysis of aptamer binding site for HCV-NS3 protease by alanine scanning mutagenesis.

Nonstructural protein 3 (NS3) of Hepatitis C virus (HCV) is a multifunctional protein and possesses protease, nucleotide triphosphatase and helicase activities. The N-terminal domain of NS3 (amino acids 1027-1218; delta NS3) has a trypsin-like protease activity and is essential for processing of viral polyprotein. Accordingly it is a potential target for anti-HCV drugs and we isolated RNA aptamers (Kd = 10 nM, Ki = 100 nM) using in vitro selection strategy. To study the interaction between delta NS3 and its aptamer, we applied alanine scanning mutagenesis and constructed seven mutant proteins at positive amino acid residues on the surface of delta NS3. Binding and inhibitory activities of the NS3 aptamer against mutant proteins were kinetically analyzed. These results clarified that especially Arg161 and Arg130 are important for interaction with the NS3 aptamer.

Base Sequence↗
PubMed2000

NMR analysis of tertiary interactions in HDV ribozymes.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems designed on the basis of the "pseudoknot" model were synthesized and their tertiary interactions were analyzed by NMR spectroscopy. Rz-1 is a cis-acting ribozyme system (the cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. Rz-2 is a trans-acting ribozyme system (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-2 was partially labeled with stable isotopes in guanosine residues of enzyme 35mer. Rz-4 is a trans-acting ribozyme system (substrate: 8mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme 53mer) which was designed by Perrotta and Been. Rz-4 has the same sequence and an extra loop closing stem IV. From 2D-NOESY and 2D-HSQC (except for Rz-4) spectra, it was suggested each ribozyme forms "pseudoknot" type structure in solution. Additionally, it was found that G38 of Rz-1, G28 and G29 of Rz-2 and Rz-4 form base-pairs. These novel base-pairs are observed in the crystal structure of a modified genomic HDV RNA. From temperature change experiment of Rz-2, the imino proton signal of G28 disappeared at 50 degrees C earlier than the other corresponding signals. Upon MgCl2 titration of Rz-2, this signal showed the largest shift.

Base Sequence↗
PubMed2000

Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides.

The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.

Bacteriophage phi X 174↗