PubMed Health⌕ Search

Biomedical subjects

T Isogai

Publications and source records attributed to T Isogai.

17 recordsLinked to original sources
PubMed2001

HSH2: a novel SH2 domain-containing adapter protein involved in tyrosine kinase signaling in hematopoietic cells.

We isolated a cDNA clone encoding a novel Src homology (SH)2 domain-containing protein of 47 kDa from a human cDNA library. As its transcript was predominantly expressed in hematopoietic cells, this gene was termed HSH2 for hematopoietic SH2 protein. This protein contains several putative protein-binding motifs, SH3-binding proline-rich regions, and phosphotyrosine sites, but lacks enzymatic motifs. In a yeast two-hybrid screen, we identified a cytokine-regulated tyrosine kinase c-FES and an activated Cdc42-associated tyrosine kinase ACK1 as HSH2 interactors. HSH2 bound c-FES via its C-terminal region as well as its N-terminal region including the SH2 domain, whereas it bound ACK1 via its N-terminal proline-rich region. Furthermore, these two kinases bound and tyrosine-phosphorylated HSH2 in mammalian cells. Hence, we postulate that HSH2 functions as an adapter protein involved in tyrosine kinase signaling, and possibly regulates cytokine signaling and cytoskeletal reorganization, in hematopoietic cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

HUNT: launch of a full-length cDNA database from the Helix Research Institute.

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.

Computational Biology↗
PubMed2001

Diverse transcriptional initiation revealed by fine, large-scale mapping of mRNA start sites.

Determination of the mRNA start site is the first step in identifying the promoter region, which is of key importance for transcriptional regulation of gene expression. The 'oligo-capping' method enabled us to introduce a sequence tag to the first base of an mRNA by replacing the cap structure of the mRNA. Using cDNA libraries made from oligo-capped mRNAs, we could identify the transcriptional start site of an individual mRNA just by sequencing the 5'-end of the cDNA. The fine mapping of transcriptional start sites was performed for 5880 mRNAs in 276 human genes. Contrary to our expectations, the majority of the genes showed a diverse distribution of transcriptional start sites. They were distributed over 61.7 bp with a standard deviation of 19.5. Our finding may reflect the dynamic nature of transcriptional initiation events of human genes in vivo.

Base Sequence↗
PubMed2001

Identification and characterization of the potential promoter regions of 1031 kinds of human genes.

To understand the mechanism of transcriptional regulation, it is essential to identify and characterize the promoter, which is located proximal to the mRNA start site. To identify the promoters from the large volumes of genomic sequences, we used mRNA start sites determined by a large-scale sequencing of the cDNA libraries constructed by the "oligo-capping" method. We aligned the mRNA start sites with the genomic sequences and retrieved adjacent sequences as potential promoter regions (PPRs) for 1031 genes. The PPR sequences were searched to determine the frequencies of major promoter elements. Among 1031 PPRs, 329 (32%) contained TATA boxes, 872 (85%) contained initiators, 999 (97%) contained GC box, and 663 (64%) contained CAAT box. Furthermore, 493 (48%) PPRs were located in CpG islands. This frequency of CpG islands was reduced in TATA(+)/Inr(+) PPRs and in the PPRs of ubiquitously expressed genes. In the PPRs of the CGM2 gene, the DRA gene, and the TM30pl genes, which showed highly colon specific expression patterns, the consensus sequences of E boxes were commonly observed. The PPRs were also useful for exploring promoter SNPs.

Base Sequence↗
PubMed2000

The molecular characterization and tissue distribution of the human cysteinyl leukotriene CysLT(2) receptor.

Cysteinyl leukotrienes (CysLTs), slow-reacting substances of anaphylaxis, are lipid mediators known to possess potent proinflammatory action. Pharmacological studies using CysLTs indicate that at least two classes of G protein-coupled receptors (GPCRs), named CysLT(1) and CysLT(2), exist; the former is sensitive and the latter is resistant to the CysLT(1) antagonists currently used to treat asthma. Although the CysLT(1) receptor gene has been recently cloned, the molecular identity of the CysLT(2) receptor has remained elusive. Here we show that the pharmacological profile of an orphan GPCR (PSEC0146) is consistent with that of the CysLT(2) receptor. In human embryonic kidney 293 cells that express the PSEC0146 cDNA, leukotriene C(4) (LTC(4)) and leukotriene D(4) (LTD(4)) induce equal increases in intracellular calcium mobilization; these increases are not affected by CysLT(1) antagonists. Additionally, [(3)H]LTC(4) specifically binds to membranes from COS-1 cells transiently transfected with PSEC0146. Large amounts of the PSEC0146 mRNA are found in human heart, placenta, spleen, and peripheral blood leukocytes but not in the lung and the trachea. Pharmacological feature and expression studies will eventually lead to a better understanding of the classification of CysLT receptors, possibly leading to a reconsideration of the pathological and physiological role of CysLTs.

Animals↗
PubMed2000

Statistical analysis of the 5' untranslated region of human mRNA using "Oligo-Capped" cDNA libraries.

We constructed 34 types of human "full-length enriched" and "5'-end enriched" cDNA libraries based on the "Oligo-Capping" method. We randomly picked and sequenced 10,000 clones from these libraries. BLAST analysis showed that about 50% of the cDNAs were identical to known genes. Among them, we selected 954 species of cDNA that should represent the entire sequence from the mRNA start sites. Compared with previously reported sequences, they were on average 45 bp longer in the 5'-end. Using these cDNA data, we statistically analyzed the sequence features of the 5'UTR. The average length of the 5'UTR was 125 bp, and there was little correlation with the corresponding mRNA length (correlation coefficient = 0.26). Of the 954 species of 5'UTR, 459 contained no in-frame terminator codon, which is against the common belief. Two hundred seventy-eight species contained at least one ATG codon upstream of the initiator ATG codon. We identified 569 upstream ATGs, in total, 63% of which adequately satisfied Kozak's criteria. These findings are contrary to the typical translation initiation model, which states that translation is initiated from the "first" ATG codon.

5' Untranslated Regions↗
PubMed2000

Prediction whether a human cDNA sequence contains initiation codon by combining statistical information and similarity with protein sequences.

MOTIVATION: In the previous works, we developed ATGpr, a computer program for predicting the fullness of a cDNA, i.e. whether it contains an initiation codon or not. Statistical information of short nucleotide fragments was fully exploited in the prediction algorithm. However, sequence similarities to known proteins, which are becoming increasingly available due to recent rapid growth of protein database, were not used in the prediction. In this work, we present a new prediction algorithm based on both statistical and similarity information, which provides better performance in sensitivity and specificity. RESULTS: We evaluated the accuracy of ATGpr for predicting fullness of cDNA sequences from human clustered ESTs of UniGene, and we obtained specificity, sensitivity, and correlation coefficient of this prediction. Specificity and sensitivity crossed at 46% over the ATGpr score threshold of 0.33 and the maximum correlation coefficient of 0.34 was obtained at this threshold. Without ATGpr we found it effective to use alignments with known proteins for predicting the fullness of cDNA sequences. That is, specificity increased monotonously as similarity (identity of the alignments) increased. Specificity was achieved greater than 80% if identity was greater than 40%. For more effective prediction of fullness of cDNA sequences we combined the similarity (identity of query sequence) with known proteins and ATGpr score. As a result, specificity became greater than 80% if identity was greater than 20%. AVAILABILITY: The prediction program, called ATGpr_ sim, is available at http://www.hri.co.jp/atgpr/ATGpr_sim.html CONTACT: nisikawa@crl.hitachi.co.jp

Amino Acid Sequence↗
PubMed2000

Preparation of sensitive and specific oligonucleotide probes tailed using terminal transferase and dITP.

An oligonucleotide probe tailed with deoxyadenosine-5'-triphosphate or deoxythymine-5'-triphosphate is detectable with high sensitivity, but has a major drawback--the tail co-hybridizes specifically to complementary sequences. This can be a problem when screening cDNA clones that contain poly(dA) sequences. While it is possible to mask the cDNA tail with unlabeled poly(dA) or poly(A) oligonucleotides, false-positive clones are still produced because complete masking of extremely long (dA) tails is difficult. As a result, only cDNA clones that have extremely long poly(dA) sequences are often obtained by hybridization screening using tailed probes. In this report, we describe an oligonucleotide probe tailed with DIG-labeled nucleotide in combination with deoxyinosine-5'-triphosphate that was highly specific and sensitive to cDNAs. Terminal deoxynucleotidyl transferase efficiently adds dI nucleotides to the 3'-end. The dI of the tails did not pair with any nucleotides under stringent hybridization so that the specificity of hybridization assays remained high without affecting the sensitivity of the test. Colony hybridization experiments demonstrated that there were very few (1 of 80 tested) false positives using this technique. Its use may increase the accuracy of cDNA screening.

DNA Nucleotidylexotransferase↗
PubMed2000

An integrated analysis and database system for full-length cDNA.

Annotation and database system of full-length cDNA sequences was developed. As the components of the system, ORF annotation system, functional annotation system based on database search results, mapping annotation system, and integrated retrieval and display system were developed. In the ORF annotation system integrated analyses using conventional tools are performed and useful retrieval interface using motif list are introduced. In the functional annotation system based on database search results, a new method that characterizes a given unknown cDNA was developed by using a profile of similarity level over words appearing in sequence database entries. In the mapping annotation system, we linked by similarity searches full-length cDNA sequences with database DNA sequences that are already mapped on chromosomes. By using these links, full-length cDNAs can be retrieved by the retrieval condition of physical mapping information. Genetic disease information mapped on the physical mapping site can also be displayed by this system. Furthermore, we constructed an integrated database system for these analyzed data, and thus enabled annotation and selection of full-length cDNAs from points of both gene function and mapping information.

Chromosome Mapping↗
PubMed1996

Fine mapping of the cystinosis gene using an integrated genetic and physical map of a region within human chromosome band 17p13.

The cystinosis gene has been reported to reside in a 3.1 cM region of chromosome 17p13 flanked by markers D17S1828 and D17S1798. We created a yeast artificial chromosome (YAC) contig between these markers and report here an integrated genetic and physical map which will aid in the identification of other genes in this area. Using one pertinent YAC clone, 898A10, we identified new polymorphic markers in the cystinosis gene region. One such marker, D17S2167, was localized by radiation hybrid analysis to within 10.2 cR8000 of D17S1828. Haplotype analysis in two separate informative families revealed recombination events which placed the cystinosis gene between markers D17S1828 and D17S2167, an area estimated to be 187-510 kb in size. This dramatic narrowing of the cystinosis gene region permits the creation of a P1 or cosmid contig across the area of interest. The ultimate cloning of the cystinosis gene should eventually reveal how a functional lysosomal transport protein is synthesized, targetted, processed, and integrated into the lysosomal membrane.

Base Sequence↗
PubMed1991

Construction of a 7-aminocephalosporanic acid (7ACA) biosynthetic operon and direct production of 7ACA in Acremonium chrysogenum.

We have used cDNA encoding D-amino acid oxidase, and genomic DNA encoding cephalosporin acylase from Fusarium solani and Pseudomonas diminuta, respectively, to construct a novel hybrid 7-aminocephalosporanic acid (7ACA) biosynthetic operon under the control of regulatory elements from the alkaline protease gene of Acremonium chrysogenum. Transformants of A. chrysogenum BC2116, a high cephalosporin-producing strain, containing this operon, synthesized and secreted low levels of 7ACA. Although the amounts are not yet commercially significant, this represents the first microbial production of 7ACA and demonstrates the feasibility of introducing new biosynthetic capabilities into industrial microorganisms by combining fungal and bacterial genes.

Acremonium↗
PubMed1991

Cloning and nucleotide sequences of the complementary and genomic DNAs for the alkaline protease from Acremonium chrysogenum.

Complementary DNA encoding Ac. chrysogenum alkaline protease (Alp) was isolated from the Ac. chrysogenum ATCC11550 cDNA library by express-blot assay. The genomic DNAs encoding Ac. chrysogenum Alp were isolated from the Ac. chrysogenum genomic DNA library using the cloned cDNA as a probe. The 3150 nucleotides of the gene were sequenced. The prepro-Alp consists of 402 amino acids and two intervening sequences are found within the coding region. The amino acid sequence of Ac. chrysogenum Alp has 57% homology to that of Aspergillus oryzae Alp. The entire cDNA, encoding Ac. chrysogenum Alp, when introducing into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium.

Acremonium↗
PubMed1991

Inhibitory effect of dietary administration of eicosapentaenoic acid on the contractions of guinea-pig tracheal smooth muscle induced by leukotriene C4 and D4.

The changes in fatty acid composition in phospholipids of guinea-pig lung parenchymal strips and trachea induced by dietary administration of eicosapentaenoic acid (EPA) were investigated as well as the resultant changes in leukotriene (LT) C4- and D4-induced contractions of guinea-pig tracheal smooth muscle. EPA levels in both parenchymal strips and trachea were significantly increased depending on the administered dose of EPA, but on the other hand, arachidonic acid levels in those preparations were not changed. Both the contractions of guinea-pig tracheal smooth muscle induced by LTC4 and D4 were significantly reduced in the EPA-treated group compared with the control group at all 3 concentrations, 10(-9), 3 x 10(-9) and 10(-8) mol/l, in the presence of 5 x 10(-5) mol/l indometacin, a cyclooxygenase inhibitor. But this significant reduction of the contraction was not recognized between these 2 groups in the presence of 10(-5) mol/l 2-(12-hydroxydodeca-5, 10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone (AA861), a 5-lipoxygenase inhibitor, or in the combined presence of 5 x 10(-5) mol/l indometacin and 10(-5) mol/l of AA861. These results suggest that: 1. a 5-lipoxygenase pathway is partly involved in the contractions of guinea-pig tracheal smooth muscle induced by LTC4 and D4 and; 2. EPA suppresses LTC4- and D4-induced contractions of guinea-pig tracheal smooth muscle through a 5-lipoxygenase pathway.

Animals↗
PubMed1990

Structure and expression of cDNA for D-amino acid oxidase active against cephalosporin C from Fusarium solani.

D-Amino acid oxidase (DAO) was extracted and purified from cultured mycelia of Fusarium solani M-0718 (FERM P-2688). The enzyme was able to oxidatively deaminate cephalosporin C to 7-beta-(5-carboxy-5-oxopentanamido)cephalosporanic acid. Ninety-eight amino acid residues of the F. solani DAO were determined by sequence analysis of 9 peptides derived from Acromobacter protease I digests of the protein. Complementary DNAs encoding F. solani DAO were isolated from the F. solani cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of the clones revealed a 1,186-nucleotide sequence with a 5'-terminal untranslated region of 41 nucleotides, an open reading frame of 1,083 nucleotides that encoded 361 amino acids, and a 3'-terminal untranslated region of 62 nucleotides. The amino acid sequence of F. solani DAO had 25% homology to that of porcine kidney DAO [EC 1.4.3.3] and 37% homology to that of Trigonopsis variabilis DAO. The constructed plasmid overproduced F. solani DAO in Escherichia coli. The recombinant DAO had almost the same molecular activity as the native DAO against cephalosporin C.

Amino Acid Sequence↗
PubMed1990

[Desquamative interstitial pneumonia-like changes in idiopathic pulmonary fibrosis].

A 61-year-old man was admitted to our hospital with fever, cough and dyspnea on exertion. The chest X-ray showed diffuse reticulo-granular infiltrates. Deterioration of clinical features and remarkable elevation of BALF lymphocytes (64.3%) suggested active interstitial pneumonia. The open lung biopsy specimen showed chronic interstitial pneumonia with DIP-like pathologic change. There was a remarkable clinical, physiological and roentgenographic improvement associated with decrease of BALF lymphocytes in response to steroid therapy. BAL is useful for monitoring disease activity and tapering steroids in patients with interstitial pneumonia who respond to steroid therapy.

Bronchoalveolar Lavage Fluid↗