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S Kuhara

Publications and source records attributed to S Kuhara.

At least 19 recordsLinked to original sources
PubMed1997

Temporal changes in expression of transforming growth factor-beta superfamily members and their receptors during bovine preodontoblast differentiation in vitro.

Transforming growth factor (TGF)-beta superfamily members and their receptors play a part in the differentiation of pulp cells into odontoblasts during reparative dentinogenesis. Bovine primary pulp-cell culture has been used as an in vitro model for proliferation and differentiation of pulp cells into preodontoblasts. To explore the molecular cascade of odontoblast differentiation, Northern blot analyses and reverse transcriptase polymerase chain reaction were here used to investigate the expression patterns of the genes for TGF-beta superfamily members: TGF-beta 1, namely bone morphogenetic protein (BMP)-4, BMP-7, activin-beta A and activin-beta B, and their type I and type II receptors, namely activin receptor-like kinase (ALK)-2 (ActR-I), ALK-3 (BMPR-IA), ALK-4 (ActR-IB), ALK-5 (T beta R-I), BMPR-II and T beta R-II, during differentiation of pulp cells into preodontoblasts in bovine adult pulp-cell culture. TGF-beta 1 and BMP-4 mRNAs were expressed from day 14 when matrix formation increased. BMP-7 mRNA was expressed only on day 28 when osteocalcin appeared. ALK-2 mRNA was increased from the beginning of the culture. ALK-3 and ALK-5 mRNAs first decreased on day 14 and increased again on day 21. T beta R-II and BMPR-II mRNAs were almost constant. These results suggest that the differentiation of pulp cells into preodontoblasts may be regulated by changes in the temporally coordinated expression pattern of TGF-beta superfamily members and their receptors, including up-regulation of transcription of TGF-beta 1, BMP-4, BMP-7, ALK-2, ALK-3, and ALK-5.

Amino Acid Sequence↗
PubMed1997

Two closely-related left-right asymmetrically expressed genes, lefty-1 and lefty-2: their distinct expression domains, chromosomal linkage and direct neuralizing activity in Xenopus embryos.

BACKGROUND: Vertebrates have numerous lateral asymmetries in the position of their organs, but the molecular basis for the determination of left-right (L-R) asymmetries remains largely unknown. TGFbeta-related genes such as lefty and nodal are L-R asymmetrically expressed in developing mouse embryos, and may be involved in L-R determination. RESULTS: We have identified two highly conserved genes, lefty-1 and lefty-2, in the mouse genome. These two genes are tightly linked on mouse chromosome 1. lefty-1 and lefty-2 are both expressed in a L-R asymmetric fashion in mouse embryos. However, the major expression domains of the two genes are different: lefty-1 expression is predominantly confied to the left side of ventral neural tube, whereas lefty-2 is strongly expressed in the lateral plate mesoderm on the left side. In embryos homozygous for the iv and inv mutation, which cause situs inversus, the expression sites of both genes are affected, either reversed or bilaterally, indicating that lefty-1 and lefty-2 are downstream of iv and inv. Although Lefty-1 and Lefty-2 prepro-proteins are not readily processed in cultured cells, BMP2-Lefty chimeric proteins can be processed to a secreted form. We have examined the activities of Lefty-1 and Lefty-2 in Xenopus embryos. In animal cap explants, Lefty-1 and Lefty-2 induce neural cells in the absence of mesoderm induction. The direct neuralizing activities of Lefty-1 and Lefty-2 thus seem remarkably similar to those of BMP antagonists such as noggin and chordin, suggesting that the action of Lefty-1 and Lefty-2 may be to locally antagonize BMP (bone morphogenic protein)-mediated signals in tissues positioned on the left side of the mouse embryos. CONCLUSION: There are two lefty genes in mice (lefty-1 and lefty-2), both of which are expressed in a L-R asymmetric fashion and are downstream of iv and inv. Lefty-1 and Lefty-2 possess direct neuralizing activity in Xenopus embryos, resembling the activities of BMP antagonists.

Amino Acid Sequence↗
PubMed1997

Application of a deductive database system to search for topological and similar three-dimensional structures in protein.

A deductive database system PACADE (Protein Atomic Coordinate Analyzer with Deductive Engine) has been developed for protein structure analysis. With this system, super-secondary structures described in logical and declarative rules can be retrieved effectively. For protein structure analysis, comparison of local structures in different proteins is a necessary mean. A function to search for similar structures has, therefore, been added to the PACADE system. We describe herein the result of searches for the same topological structures and three-dimensionally similar ones. A user of PACADE can select these two levels of similarity by changing parameters. This function enables the inference system to retrieve similar structures, according to the restraints of variables defined by the user. Similar super-secondary structures among proteins can be searched for automatically, which is useful for protein structure analysis. The retrieved similar super-secondary structures can serve as criteria for protein spatial alignment.

Algorithms↗
PubMed1997

Expression of TGF-beta superfamily receptors in dental pulp.

Transforming growth factor-beta (TGF-beta) superfamily members and their cell-surface receptors may play inductive and/or regulatory roles in tooth development and repair. It will be important to identify the complete set of TGF-beta superfamily receptors, to examine their temporal and spatial localization during tooth development, and to elucidate the cascade of molecular events of tooth formation induced by the TGF-beta superfamily. In this report, we have cloned the cDNAs encoding potential receptors for TGF-beta superfamily members in rat incisor pulp and bovine adult pulp which are regarded as embryonic and adult pulp, respectively. We analyzed poly (A)+ RNA from rat incisor pulp and bovine adult pulp by reverse-transcriptase/polymerase chain-reaction (RT-PCR), using a degenerate primers corresponding to the most conserved amino acid sequences in the intracellular serine/threonine kinase of type I or type II like kinase-1 (ALK-1), ALK-2, ALK-3 (bone morphogenetic protein receptor type IA, BMPR-IA), ALK-4 (B1), ALK-5, ALK-6 (BMPR-IB), and BMPR-II (BMP type II receptor) was found to be in dental pulp. Northern blot analysis further detected TGF-beta type II receptor (T beta R-II) mRNA transcript in addition to the above-identified receptors. These results provide the first evidence of multiple type I and type II receptors for TGF-beta s, activins, and BMPs expressed in embryonic and adult pulp, implicating diverse function in tooth development and pulp tissue repair.

Activin Receptors↗
PubMed1997

Extraction of substructures of proteins essential to their biological functions by a data mining technique.

Correlation between the sequential, structural, and functional features of proteins is one of the most important open questions in the field of molecular biology. To this problem, we apply a technique known as data mining for discovering associations across protein sequence, structure, and function. We were able to find various association rules on the substructures essential to some protein functions. Moreover, structure-structure associations were found between proteins having different functions. The results suggest that data mining might be a powerful tool in protein analysis.

Algorithms↗
PubMed1997

Finding association rules on heterogeneous genome data.

A novel approach for discovery of knowledge from genome data, which has been recently watched with interest in the research area of database, is applied to finding unified rules spreading over sequence, structure, and function of protein. As the result of experiments using data extracted from PDB, SWISS-PROT, and PROSITE, some association rules stating sequential/structural/functional aspects of two kinds of endopeptidases were found.

Computer Simulation↗
PubMed1996

Glycine-15 in the bend between two alpha-helices can explain the thermostability of DNA binding protein HU from Bacillus stearothermophilus.

On the basis of sequence comparison of thermophilic and mesophilic DNA binding protein HUs, Bacillus stearothermophilus DNA binding protein HU (BstHU) seems to gain thermostability with a change in amino acid residues present on the molecular surface. To evaluate the contribution of exchange of each amino acid to the thermostability of BstHU, we constructed three mutants, BstHU-T13A (Thr13 to Ala), BstHU-G15E (Gly15 to Glu), and BstHU-T33L (Thr33 to Leu), in which the amino acids in BstHU were changed to the corresponding ones in Bacillus subtilis DNA binding protein HU (BsuHU). Stability of the mutant proteins was determined from thermal-denaturation curves. Replacement of Gly15 located in the turn region between alpha 1 and alpha 2 helices (HTH motif), with Glu (BstHU-G15E), resulted in a decrease in thermostability, and the Tm value was 54.0 degrees C compared to the Tm value of 63.9 degrees C for BstHU. The mutants, BstHU-T13A and BstHU-T33L, were, by contrast, slightly more stable (Tm values of 67.0 and 65.6 degrees C for BstHU-T13A and BstHU-T33L, respectively) than the wild type. We then generated the BsuHU mutant protein BsuHU-E15G, where Glu15 in BsuHU was in turn replaced by Gly, and we analyzed the thermostability. This substitution clearly enhanced the melting temperature by 11.8 degrees C (Tm value: 60.4 degrees C for BsuHU-E15G) compared to the value for BsuHU (Tm: 48.6 degrees C). Thus, Gly15 in the HTH motif of BstHU has an important role in the thermostability of BstHU. Characterization of the structure of the BstHU-G15E by 1H-NMR analysis showed that solvent accessibility of amide proton of Ala21 in the mutant was significantly increased compared with that of wild type, which means that the structure of the HTH motif in the N-terminal region in the mutant was changed to a more open conformation, thereby avoiding the interaction of Ala21 with either Ser17 by hydrogen bond or Ala11 by hydrophobic interaction.

Bacterial Proteins↗
PubMed1996

In vivo rapid magnetic field measurement and shimming using single scan differential phase mapping.

In vivo shimming is a powerful tool for alleviating subject-dependent magnetic field distortions in MRI, although the usually lengthy procedure has limited its use. A magnetic field measurement method called single scan differential phase mapping that can reduce the measurement time of conventional methods by more than half when used with multiple gradient-recalled echo imaging was developed. An automatic high-order shimming using the present method was implemented in a commercial MRI system. In vivo field inhomogeneity features and the shimming performances were investigated. The method was found to be useful for improving the quality of echo-planar images.

Abdomen↗
PubMed1995

Differences in MHC class I self peptide repertoires among HLA-A2 subtypes.

To investigate how single amino acid substitutions in MHC class I molecules affect differences in peptide repertoires, we eluted and sequenced the naturally processed peptides from three HLA-A2 subtypes (HLA-A*0204, -A*0206, and -A*0207) that differ by a single amino acid residue substitution each with HLA-A*0201 at the floor of the binding groove. Allele-specific peptide motifs for each HLA-A2 subtype substantially differed from that of HLA-A*0201 in the dominant anchor residues. The relative signal intensities for 18 self peptides, determined by mass spectrometry, precisely reflected these peptide motifs. Some overlapping peptides were isolated from both HLA-A*0201 and a single HLA-A2 variant, but no peptide was ubiquitously found across all variants. To rationalize the differences in peptide motifs, possible conformations of each allele were computer modeled by energy minimization calculations based on the reported crystal structure of HLA-A*0201. According to our models, the differences in peptide motifs could be explained by substituted-residue-driven conformational changes for each MHC-peptide complex. These results demonstrate the fine differences between HLA-A2 subtype self peptide repertoires and contribute to the prediction of antigenic peptides.

Amino Acid Sequence↗
PubMed1995

BONSAI Garden: parallel knowledge discovery system for amino acid sequences.

We have developed a machine discovery system BONSAI which receives positive and negative examples as inputs and produces as a hypothesis a pair of a decision tree over regular patterns and an alphabet indexing. This system has succeeded in discovering reasonable knowledge on transmembrane domain sequences and signal peptide sequences by computer experiments. However, when several kinds of sequences are mixed in the data, it does not seem reasonable for a single BONSAI system to find a hypothesis of a reasonably small size with high accuracy. For this purpose, we have designed a system BONSAI Garden, in which several BONSAI's and a program called Gardener run over a network in parallel, to partition the data into some number of classes together with hypotheses explaining these classes accurately.

Algorithms↗
PubMed1994

A yeast gene necessary for bud-site selection encodes a protein similar to insulin-degrading enzymes.

Cells of the yeast Saccharomyces cerevisiae choose bud sites in a non-random spatial pattern that depends on mating type: axial for haploid cells and bipolar for a/alpha diploid cells. We identified a mutant yeast, axl 1, in which the budding pattern is altered from axial to bipolar. Expression of the AXL1 gene is repressed in a/alpha diploid cells. With the ectopic expression of AXL1, a/alpha cells exhibited an axial budding pattern, thus AXL1 is a key morphological determinant that distinguishes the budding pattern of haploid cells from that of a/alpha diploid cells. AXL1 encodes a protein similar in sequence of the human and Drosophila insulin-degrading enzymes and to the Escherichia coli ptr gene product. The axial budding pattern might result from degradation of a target protein by the putative Axl1 protease.

Amino Acid Sequence↗
PubMed1994

Interfacial kinetic reaction of human 5-lipoxygenase.

The kinetics of human 5-lipoxygenase were investigated in the presence of Tween 20 using a continuous spectrophotometric assay. Using the mixture at a constant molar ratio of arachidonate/Tween 20 at pH 8.0, the steady-state velocity on a varied arachidonate concentration did not follow simple Michaelis-Menten-type kinetics and double-reciprocal plot analysis gave hyperbolic curves. However, by introducing the concept of a local pH change, it was possible to analyze the kinetics as simple Michaelis-Menten type. The concept of a local pH change implies that when utilizing an acidic and amphiphilic substance as a substrate, such as arachidonate, the medium around the substrate is acidified with an increased concentration of substrate. This concept was explained rationally by two experiments. Consequently, the data were transformed according to a local pH change and analyzed according to a dual phospholipid model as has been proposed for phospholipase A2 [Hendrickson, H. S. and Dennis, E. A. (1984) Kinetic analysis of the dual phospholipid model for phosphalipase A2, J. Biol. Chem. 259, 5734-5739]. It is concluded that 5-lipoxygenase performs an interfacial reaction in the arachidonate/Tween 20 mixed micelles in the same manner as phospholipase A2. The values of Km were almost constant (about 0.07 molar fraction), even when arachidonate molar ratios were changed in the surface of the mixed micelles. The values for Ks (the association constant of the enzyme to the micelle interface) ranged over 0.21-0.48 microM. The Vmax was 25.76 mumol.min-1.mg-1. This concept of a local pH change could be used extensively with enzymes which utilize both amphiphilic and acidic substances as substrates.

Arachidonate 5-Lipoxygenase↗
PubMed1994

Molecular cloning of the gene encoding a highly expressed protein in SFL1 gene-disrupted flocculating yeast.

We identified a yeast gene encoding Flocculent Specific Protein (FSP) produced excessively in the SFL1 gene-disrupted flocculent strain. The sequenced gene encodes a 430 amino acid protein and is mainly composed of multiple repeats of Ser-Asn-Asn-X-Asp-Ser-Tyr-Gly. The FSP gene disruption of the flocculent strain decreased the degree of flocculation, so FSP may be one factor concerned with yeast flocculation. A gene database search indicated that the FSP gene is identical with the DDR48 gene.

Amino Acid Sequence↗
PubMed1994

Streptomyces ATP nucleotide 3'-pyrophosphokinase-gene cloning and sequence analysis.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-beta, gamma-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.

Amino Acid Sequence↗
PubMed1993

Sequence-tagged NotI sites of human chromosome 21: sequence analysis and mapping.

We isolated and analyzed 19 NotI site-containing clones specific for human chromosome 21. The overall process consisted of selective isolation of NotI site-containing clones from flow-sorted chromosome 21 libraries, selection of independent clones by their restriction patterns and nucleotide sequences, and assignment of the clones to chromosome 21. Sequence analysis showed that the regions around the NotI sites had features typical of CpG islands, such as extremely high GC content, high-frequency appearance of CpG dinucleotide sequences, and lack of methylation of these CpGs. PCR conditions for these extremely G + C-rich templates were optimized to establish these NotI sites as sequence-tagged sites.

Base Sequence↗
PubMed1993

A deductive database system PACADE for analyzing 3-D and secondary structures of protein.

We have developed a deductive database system PACADE for analyzing 3-D and secondary structures of protein. The PACADE system consists of a relational database created from Protein Data Bank and a deductive engine DEE based on logic programming. It has the following features: (1) The system has an inference mechanism. This means by which users can easily write and check biological hypotheses using logical and declarative rules instead of procedural programs. (2) The relational database of the PACADE system stores data on both 3-D and secondary structures of protein. The integration of this two level structure makes feasible an abstract representation of the protein structure. We describe herein the design, functions, and implementation of this PACADE system.

Computer Graphics↗
PubMed1993

Sporulation-inhibitory gene in pock-forming plasmid pSA1.1 of Streptomyces azureus.

A plasmid, pSA1.1, of Streptomyces azureus elicited conjugative pocks and inhibited the sporulation of its host mycelia. pSA1.1 was studied with a kanamycin resistance gene derived from S. kanamyceticus as a selective marker. The deletion analysis and sequencing of the derivative plasmid found an open reading frame (ORF909b) that was involved in the sporulation-inhibitory function of pSA1.1 and in pock formation. The nucleotide sequences of ORF909b were compared with those of the genes registered in GenBank, but no similarity was found. However, the predicted amino acid sequence of ORF909b showed a significantly high similarity with that of the spoIIIE gene of Bacillus subtilis. This detected gene might be a new sporulation-regulatory gene in streptomycetes.

Amino Acid Sequence↗