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Y Sakaki

Publications and source records attributed to Y Sakaki.

At least 19 recordsLinked to original sources
PubMed2006

CSF1 gene associated with aggressive periodontitis in the Japanese population.

Aggressive periodontitis (AgP) is characterized by the early onset of the rapid and progressive destruction of the alveolar bone. We investigated the correlation of single nucleotide polymorphisms (SNPs) in candidate genes with AgP in the Japanese population in order to determine the genetic risk factors for this complex disease. Among 11 genes related to bone formation and resorption, 43 known SNPs were tested in 98 case and 88 control samples for association with AgP by using SNP genotyping techniques. Among these, three polymorphisms located in the colony stimulating factor 1 (CSF1) gene showed a positive association with AgP. This is the first case of an association between a CSF1 polymorphism and a human disease.

Adolescent↗
PubMed2006

FLT3 is fused to ETV6 in a myeloproliferative disorder with hypereosinophilia and a t(12;13)(p13;q12) translocation.

The FMS-like tyrosine kinase 3 (FLT3) gene, belonging to the receptor tyrosine kinase (TK) subclass III family, plays an important role in normal hematopoiesis and is one of the most frequently mutated genes in hematologic malignancies as well as an attractive target for directed inhibition. Activating mutations of this gene, including internal tandem duplication in the juxtamembrane (JM) domain and point mutations in the TK domain, are found in approximately one-third of patients with acute myeloid leukemia and in a smaller subset of patients with acute lymphoblastic leukemia. We report here that FLT3 may contribute to leukemogenesis in a patient with myeloproliferative disorder and a t(12;13)(p13;q12) translocation through generating a fusion gene with the ETS variant gene 6 (ETV6) gene. ETV6 has been reported to fuse to various partner genes, including TK and transcription factors. Both ETV6/FLT3 and reciprocal FLT3/ETV6 transcripts were detected in the patient mRNA by reverse transcriptase-polymerase chain reaction. At the protein level, however, only ETV6/FLT3 products were expressed. Among them, one retains the helix-loop-helix (HLH) oligomerization domain of ETV6 and the JM as well as TK domain of FLT3. FLT3 receptor in leukemic cells might be inappropriately activated through dimerization by HLH domain of ETV6, which consequently interfered with proliferation and differentiation of hematopoietic cells.

Aged↗
PubMed2006

Estimation of the species-specific mutation rates at the DRB1 locus in humans and chimpanzee.

To estimate the species-specific mutation rates at the DRB1 locus in humans and chimpanzee, we analyzed the nucleotide sequence of a 37.6-kb chimpanzee chromosomal segment containing the entire Patr-DRB1*0701 allele and the flanking nongenic region and we compared it with two corresponding human sequences containing the HLA-DRB1*070101 allele using the sequence of HLA-DRB1*04011 as an outgroup. Because the allelic pair of HLA-DRB1*070101 and Patr-DRB1*0701 shows the lowest number of substitutions between the two species, it appears that these sequences diverged close to the time of the humans-chimpanzee divergence (6 million years ago). Alignment of the nucleotide sequences for HLA-DRB1*070101 and Patr-DRB1*0701 alleles showed that they share a high degree of similarity, suggesting that the studied chromosomal segments with these sequences have not been subjected to recombination since the humans-chimpanzee divergence. Comparison of the flanking 10.6 kb of nongenic sequences revealed an average of 41.5 and 83 single nucleotide substitutions in humans and chimpanzee, respectively. Thus, the species-specific nucleotide substitution rates in the flanking nongenic region were estimated to be 6.53 x 10(-10) and 1.31 x 10(-9) per site per year in humans and chimpanzee, respectively. Unexpectedly, the estimated rate in humans was twofold lower than in chimpanzee (P < 10(-3), Tajima's relative rate test) and lower than the average substitution rate in the human genome. Because the nucleotide substitution rate in nongenic regions free from selection is expected to be equal to the mutation rate, the estimated substitution rate should correspond to the species-specific mutation rate at the DRB1 locus. Our results strongly suggest that the mutation rate at DRB1 locus differs among species.

Alleles↗
PubMed2006

Evaluation of RANK/RANKL/OPG gene polymorphisms in aggressive periodontitis.

BACKGROUND AND OBJECTIVE: Aggressive periodontitis (AgP) is a specific type of periodontal disease that is characterized by rapid attachment loss and bone destruction. While attempting to identify genetic polymorphisms associated with AgP, previous research has focused on candidate genes that may be involved in immune responses to microbial infections. In this study, the focus was on single nucleotide polymorphisms (SNPs) in the key mediators of osteoclast differentiation and activation, which involve receptor activator of nuclear factor-kappaB (RANK), RANK ligand (RANKL) and osteoprotegrin (OPG), in the Japanese population. The aim of this study was to evaluate the association of RANK/RANKL/OPG gene polymorphisms with AgP in the Japanese population. MATERIAL AND METHODS: We examined 99 patients with AgP and 89 controls from the Japanese population to explore the possibility of RANK/RANKL/OPG loci as candidate regions associated with the disease. All exons and relevant exon-intron boundaries of these three candidate genes were amplified by polymerase chain reaction (PCR) using 19 primers, followed by direct sequencing. The polymorphisms were identified by comparing the sequences obtained from 48 subjects. RESULTS: We identified 27 SNPs in RANK, including 10 novel SNPs and seven SNPs each in both RANKL and OPG. A pairwise linkage disequilibrium analysis using the r2 statistic showed that some SNP pairs from the three loci are in tight linkage disequilibrium. CONCLUSION: An association analysis with allelotypes showed that SNPs identified in the RANK/RANKL/OPG genes have no significant association with AgP in the Japanese population.

Acute Disease↗
PubMed2004

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Animals↗
PubMed2003

DIGIT: a novel gene finding program by combining gene-finders.

We have developed a general purpose algorithm which finds genes by combining plural existing gene-finders. The algorithm has been implemented into a novel gene-finder named DIGIT. An outline of the algorithm is as follows. First, existing gene-finders are applied to an uncharacterized genomic sequence (input sequence). Next, DIGIT produces all possible exons from the results of gene-finders, and assigns them their exon types, reading frames and exon scores. Finally, DIGIT searches a set of exons whose additive score is maximized under their reading frame constraints. Bayesian procedure and a hidden Markov model are used to infer exon scores and search the exon set, respectively. We have designed DIGIT so as to combine the results of FGENESH, GENSCAN and HMMgene, and have assessed its prediction accuracy by using recently compiled benchmark data sets. For all data sets, DIGIT successfully discarded many false-positive exons predicted by individual gene-finders and yielded remarkable improvements in sensitivity and specificity at the gene level compared with the best gene level accuracies achieved by any single gene-finder.

Algorithms↗
PubMed2001

Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites.

Streptomyces avermitilis is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites, one of which, avermectin, is commercially important in human and veterinary medicine. The major interest in this genus Streptomyces is the diversity of its production of secondary metabolites as an industrial microorganism. A major factor in its prominence as a producer of the variety of secondary metabolites is its possession of several metabolic pathways for biosynthesis. Here we report sequence analysis of S. avermitilis, covering 99% of its genome. At least 8.7 million base pairs exist in the linear chromosome; this is the largest bacterial genome sequence, and it provides insights into the intrinsic diversity of the production of the secondary metabolites of Streptomyces. Twenty-five kinds of secondary metabolite gene clusters were found in the genome of S. avermitilis. Four of them are concerned with the biosyntheses of melanin pigments, in which two clusters encode tyrosinase and its cofactor, another two encode an ochronotic pigment derived from homogentiginic acid, and another polyketide-derived melanin. The gene clusters for carotenoid and siderophore biosyntheses are composed of seven and five genes, respectively. There are eight kinds of gene clusters for type-I polyketide compound biosyntheses, and two clusters are involved in the biosyntheses of type-II polyketide-derived compounds. Furthermore, a polyketide synthase that resembles phloroglucinol synthase was detected. Eight clusters are involved in the biosyntheses of peptide compounds that are synthesized by nonribosomal peptide synthetases. These secondary metabolite clusters are widely located in the genome but half of them are near both ends of the genome. The total length of these clusters occupies about 6.4% of the genome.

Base Sequence↗
PubMed2001

Differential display analysis of mutants for the transcription factor Pdr1p regulating multidrug resistance in the budding yeast.

The transcription factor Pdr1p recognizes Pdr1p/Pdr3p-response element (PDRE) to activate genes involved in multidrug resistance of the budding yeast. To identify novel targets of Pdr1p, we compared transcriptomes among the yeast cells bearing wild, disrupted and gain-of-function alleles of PDR1 using a high-throughput fluorescent differential display PCR. Consequently, we identified 20 transcripts apparently regulated by Pdr1p, which are derived from well-known target genes as well as those that have never been described in the context of drug resistance. Intriguingly, among the latter, a previously unrecognized gene bearing a small putative open reading frame preceded by a functional PDRE was found.

Base Sequence↗
PubMed2001

Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells.

A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using 'module-shuffling primers' (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay.

Animals↗
PubMed2001

Discovery of a novel compound: insight into mechanisms for acrylamide-induced axonopathy and colchicine-induced apoptotic neuronal cell death.

The exposure of humans and experimental animals to certain industrial toxins such as acrylamide is known to cause nerve damage classified as axonopathy, but the mechanisms involved are poorly understood. Here we show that acrylamide induces morphological changes and tyrosine phosphorylation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), a member of the FAK subfamily, in human differentiating neuroblastoma SH-SY5Y cells. Furthermore, we identified a novel molecule designated 'compound-1' that inhibits the morphological and biochemical events. Daily oral administrations of the compound also effectively alleviated behavioral deficits in animals elicited by acrylamide in inclined plane testing, landing foot spread testing and rota-rod performance testing. The compound also effectively inhibited the biological and biochemical responses caused by another axonopathy inducer, colchicine, including tyrosine phosphorylation of Pyk2, formation of an 85-kDa poly(ADP-ribose)polymerase (PARP) fragment and apoptosis-associated induction of the NAPOR gene as well as neuronal cell death. Our findings not only provide insight into FAK and Pyk2 functions in neuronal cells, but may also be important in the development of therapeutic agents for peripheral neuropathy and neurodegeneration.

Acrylamides↗
PubMed2001

Isolation of hNap1BP which interacts with human Nap1 (NCKAP1) whose expression is down-regulated in Alzheimer's disease.

We previously reported the isolation of a novel apoptosis-related gene, human Nap1 (HGMW-approved symbol NCKAP1), the expression of which was strongly down-regulated in sporadic Alzheimer's disease (AD). Human Nap1 proved to be an orthologue of rat Nap1 which binds to the adaptor molecule Nck in signal transduction. In order to further elucidate the function of human Nap1, we performed yeast two-hybrid screening. As a result of screening, we discovered a protein designated hNap1BP (human Nap1 binding protein) which is a member of the tyrosine kinase-binding protein family. In addition, hNap1BP bound to the SH3 domain of c-Abl and Nck. hNap1BP is expressed ubiquitously in various tissues like human Nap1, and intriguingly these genes are co-expressed in hippocampus and cerebral cortex in mouse brain where AD pathological features are strongly evident. Further functional analysis of hNap1BP may clarify its contribution to AD pathology.

3T3 Cells↗
PubMed2001

A comprehensive two-hybrid analysis to explore the yeast protein interactome.

Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

Fungal Proteins↗
PubMed2001

Budding yeast GCN1 binds the GI domain to activate the eIF2alpha kinase GCN2.

When starved for a single amino acid, the budding yeast Saccharomyces cerevisiae activates the eukaryotic initiation factor 2alpha (eIF2alpha) kinase GCN2 in a GCN1-dependent manner. Phosphorylated eIF2alpha inhibits general translation but selectively derepresses the synthesis of the transcription factor GCN4, which leads to coordinated induction of genes involved in biosynthesis of various amino acids, a phenomenon called general control response. We recently demonstrated that this response requires binding of GCN1 to the GI domain occurring at the N terminus of GCN2 (Kubota, H., Sakaki, Y., and Ito, T. (2000) J. Biol. Chem. 275, 20243-20246). Here we provide the first evidence for the involvement of GCN1-GCN2 interaction in activation of GCN2 per se. We identified a C-terminal segment of GCN1 sufficient to bind the GI domain and used a novel dual bait two-hybrid method to identify mutations rendering GCN1 incapable of interacting with GCN2. The yeast bearing such an allele, gcn1-F2291L, fails to display derepression of GCN4 translation and hence general control response, as does a GI domain mutant, gcn2-Y74A, defective in association with GCN1. Furthermore, we demonstrated that phosphorylation of eIF2alpha is impaired in both mutants. Since GCN2 is the sole eIF2alpha kinase in yeast, these findings indicate a critical role of GCN1-GCN2 interaction in activation of the kinase in vivo.

Amino Acid Sequence↗
PubMed2001

A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

Chromosomes, Artificial, Bacterial↗
PubMed2001

Crucial role of type 1, but not type 3, inositol 1,4,5-trisphosphate (IP(3)) receptors in IP(3)-induced Ca(2+) release, capacitative Ca(2+) entry, and proliferation of A7r5 vascular smooth muscle cells.

Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) ([Ca(2+)](i)) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP(3)) mediates vasopressin (VP)-evoked Ca(2+) release from intracellular stores and showed that types 1 (IP(3)R(1)) and 3 (IP(3)R(3)) IP(3) receptors were expressed. Using antisera selective for IP(3)R(1) or IP(3)R(3) and another that interacted equally well with both subtypes, together with membranes from SF:9 cells expressing only single IP(3)R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP(3)R(1) and 19% IP(3)R(3). To elucidate the contributions of IP(3)R(1) and IP(3)R(3) to Ca(2+) signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP(3)R subtypes were selected. Mild inhibition of IP(3)R(1) (71+/-8% of control level) slightly attenuated the IP(3)-evoked Ca(2+) release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca(2+) entry (CCE) and proliferation. Moderate inhibition (34+/-6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP(3)R(3) expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP(3)R(1) and IP(3)R(3). A high dose of heparin had a similar effect to complete inhibition of IP(3)R(1) expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP(3)R(1), but not IP(3)R(3), is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.

Animals↗
PubMed2001

Entrainment of the circadian clock in the liver by feeding.

Circadian rhythms of behavior are driven by oscillators in the brain that are coupled to the environmental light cycle. Circadian rhythms of gene expression occur widely in peripheral organs. It is unclear how these multiple rhythms are coupled together to form a coherent system. To study such coupling, we investigated the effects of cycles of food availability (which exert powerful entraining effects on behavior) on the rhythms of gene expression in the liver, lung, and suprachiasmatic nucleus (SCN). We used a transgenic rat model whose tissues express luciferase in vitro. Although rhythmicity in the SCN remained phase-locked to the light-dark cycle, restricted feeding rapidly entrained the liver, shifting its rhythm by 10 hours within 2 days. Our results demonstrate that feeding cycles can entrain the liver independently of the SCN and the light cycle, and they suggest the need to reexamine the mammalian circadian hierarchy. They also raise the possibility that peripheral circadian oscillators like those in the liver may be coupled to the SCN primarily through rhythmic behavior, such as feeding.

Animals↗
PubMed2001

A novel superoxide-producing NAD(P)H oxidase in kidney.

During phagocytosis, gp91(phox), the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis. Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits 39% identity to gp91(phox) with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase. The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA. The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys. In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells. In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth. The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney.

Adult↗