Biomedical subjects

S Sugano

Publications and source records attributed to S Sugano.

At least 55 records · Page 3Linked to original sources

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

Comparative PCR: a simple and sensitive method for quantifying low-abundance mRNA species.

To measure low-abundance messenger RNA species comparatively, we developed a simple and highly sensitive quantification method designated comparative PCR. Messenger RNAs from two samples were converted into cDNAs with modified oligo(dT) primers (designated RT primers) containing a sample-specific sequence and a common sequence. After equal amounts of the cDNAs were mixed together, a target gene was amplified by competitive PCR with additional primers: a gene-specific primer and a primer consisting of the common sequence of the RT primers. The amplified products were visualized by the final PCR, designated fluorescence PCR, with an additional three primers: two different fluorescence-labeled primers consisting of the sample-specific sequence within the RT primers and a nested gene-specific primer. Expression levels of the target gene in the two samples were measured by calculating ratios of two different fluorescence intensities. We could quantify 0.1-0.3 copies of the target mRNA per cell from only 0.5 ng of poly(A)(+) RNA for a single detection. This system should be useful for sensitive measurement of scarce transcripts from small samples with a limited amount of RNA such as biopsy specimens.

Animals↗

PubMed2000

A novel gene, DSCR5, from the distal Down syndrome critical region on chromosome 21q22.2.

Based on a detailed sequence of the distal Down syndrome critical region (DSCR), we predicted and molecularly cloned a novel gene, designated DSCR5. We determined the sequences of expressed sequence tags (ESTs) that almost matched the predicted cDNA sequence of DSCR5. Northern blot analysis showed that DSCR5 is expressed in several tissues including the liver, skeletal muscle, heart, pancreas and testis. To determine the 5'-end of DSCR5, the oligo-capping method was employed. Combining the EST sequence data and that from the oligo-capping experiments, we obtained the full-length cDNA sequence of DSCR5. DSCR5 had at least four types of alternatively spliced variants. According to the number of exons, they could be classified into two subtypes: DSCR5alpha and DSCR5beta. DSCR5alpha includes three splice variant subtypes, DSCR5alpha1, alpha2 and alpha3, which each has different first non-coding exon. In addition, the most abundantly isolated form, DSCR5alpha1, shows microheterogeneity of the mRNA start site. Comparison of the sequences between the predicted cDNA and the molecularly cloned cDNA revealed that the computer programs had limited validity to correctly predict the terminal exons. Thus, molecular cloning should always be required to complement the inadequacy of the computer predictions.

Alternative Splicing↗

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

Hunting the subset-specific genes of neuroblastoma: expression profiling and differential screening of the full-length-enriched oligo-capping cDNA libraries.

BACKGROUND: Neuroblastoma (NBL) has a distinct nature in different prognostic subgroups. PROCEDURE: To understand the molecular mechanism of NBL's genesis and biology as well as that of the neural crest development, we constructed full-length-enriched cDNA libraries by an oligo-capping method from two different subsets of primary NBL, one with favorable biology and the other with MYCN amplification. RESULTS: Sequencing analysis of these libraries revealed that the expression profile was markedly different between both subsets. To identify the genes differentially expressed between the subsets, semi-quantitative RT-PCR analyses are proceeding. CONCLUSION: So far, 54 transcripts have been found to be expressed at high levels in favorable NBLs, and significantly at low levels in unfavorable NBLs.

Gene Expression Regulation, Neoplastic↗

PubMed2000

Association between favorable neuroblastoma and high expression of the novel metalloproteinase gene, nbla3145/XCE, cloned by differential screening of the full-length-enriched oligo-capping neuroblastoma cDNA libraries.

BACKGROUND: The biology of neuroblastoma (NBL) is at least partly regulated by neurotrophic factors and their receptors. PROCEDURE: To identify novel NBL-related genes that affect growth, differentiation, and programmed cell death of the tumor, we constructed full-length-enriched cDNA libraries by oligo-capping method. RESULTS: Semi-quantitative and quantitative real-time RT-PCR showed that the nbla3145 gene was significantly highly expressed in favorable subset of NBL. The nucleo-tide sequence of nbla3145 revealed that it was a novel member of metalloproteinase, endothelin-converting enzyme, and was the same gene recently reported as XCE. We also cloned nbla3145beta which was a novel truncated form of nbla3145 (or nbla3145alpha). CONCLUSIONS: Our results suggested that nbla3145/XCE plays an important role in regulating growth and differentiation of NBL.

Gene Expression Regulation, Neoplastic↗

PubMed2000

cDNA cloning, expression profile, and genomic structure of human and mouse RNF10/Rnf 10 genes, encoding a novel RING finger protein.

RING finger (C3HC4-type zinc finger) is a variant zinc finger motif present in a new family of proteins including transcription regulators. A new member of the RING finger protein family was identified through a mouse expressed sequence tag (EST) database search, and its full-length cDNA was isolated from a mouse brain full length-enriched cDNA library. The gene was designated as Rnf10, for RING finger protein 10. The cDNA clone consists of 3110 nucleotides and encodes an open reading frame (ORF) of an 804-amino acid protein. A database search revealed that human KIAA0262 protein (accession number, D87451) has strong homology to mouse Rnf10. To confirm that mouse Rnf10 is the homolog or an isolog of human KIAA0262, a human RNF10 cDNA was cloned in our hands from a fetal brain cDNA pool. The newly isolated cDNA contained an ORF for 811 amino acids which had almost identical structure to mouse Rnf10 protein, indicating that the human ORF codes for RNF10 protein. This finding was also supported by comparative chromosome mapping in which both genes were localized in a conserved linkage homology region between mouse and human. Comparison of the RNF10 and KIAA0262 proteins revealed that both were transcribed from the same gene and that the longer RNF10 ORF would be the authentic form. The complete genomic organization of RNF10 was determined to consist of 17 exons spanning at least 40kb in the genome.

Amino Acid Sequence↗

PubMed2000

Isolation and characterization of a human cDNA encoding a protein homologous to the 7.2-kDa protein (subunit X) of bovine ubiquinol-cytochrome C reductase.

Through large-scale sequencing of clones randomly selected from a library of human cDNAs, we have isolated a novel human gene termed hUQCR10. Its open reading frame encodes 63 amino acids that share 88.5% identity with the sequence of bovine ubiquinol-cytochrome C reductase 7.2-kDa protein (subunit X). A single 0.6-kb transcript was expressed in all human tissues examined, but was particularly abundant in heart and skeletal muscle, tissues that consume a large amount of oxygen. The gene product therefore may play a significant role in the cellular respiratory system. In support of this hypothesis, our immunohistochemical analysis revealed that the hUQCR10 protein is located in mitochondria. A homology search using computer programs determined the chromosomal localization of the gene at 22q12.

Animals↗

PubMed2000

Sequence analysis, gene expression, and chromosomal assignment of mouse Borg4 gene and its human orthologue.

The Borg (binder of Rho GTPases) family proteins interact with CDC42 and TC10 in a guanosine triphosphate (GTP)-dependent manner. We have isolated a full-length cDNA of the mouse Borg4 gene, which is a member of this family. Sequence analysis revealed that this gene encoded a putative 349-amino acid protein. By reverse transcription--coupled polymerase chain reaction (RT-PCR) analysis, we observed that Borg4 was expressed ubiquitously in adult tissues. Additionally, we determined the entire cDNA sequence of the putative human Borg4 orthologue. By fluorescence in situ hybridization, mouse Borg4 and the putative human orthologue have been assigned to mouse chromosome 11E and human chromosome 17q24-25, which has been described as syntenic to the mouse region.

Adaptor Proteins, Signal Transducing↗

PubMed2000

All in good time: the Arabidopsis circadian clock.

Biological time-keeping mechanisms have fascinated researchers since the movement of leaves with a daily rhythm was first described >270 years ago. The circadian clock confers a approximately 24-hour rhythm on a range of processes including leaf movements and the expression of some genes. Molecular mechanisms and components underlying clock function have been described in recent years for several animal and prokaryotic organisms, and those of plants are beginning to be characterized. The emerging model of the Arabidopsis clock has mechanistic parallels with the clocks of other model organisms, which consist of positive and negative feedback loops, but the molecular components appear to be unique to plants.

Animals↗

PubMed2000

CART classification of human 5' UTR sequences.

A nonredundant database of 2312 full-length human 5'-untranslated regions (UTRs) was carefully prepared using state-of-the-art experimental and computational technologies. A comprehensive computational analysis of this data was conducted for characterizing the 5' UTR features. Classification and regression tree (CART) analysis was used to classify the data into three distinct classes. Class I consists of mRNAs that are believed to be poorly translated with long 5' UTRs filled with potential inhibitory features. Class II consists of terminal oligopyrimidine tract (TOP) mRNAs that are regulated in a growth-dependent manner, and class III consists of mRNAs with favorable 5' UTR features that may help efficient translation. The most accurate tree we found has 92.5% classification accuracy as estimated by cross validation. The classification model included the presence of TOP, a secondary structure, 5' UTR length, and the presence of upstream AUGs (uAUGs) as the most relevant variables. The present classification and characterization of the 5' UTRs provide precious information for better understanding the translational regulation of human mRNAs. Furthermore, this database and classification can help people build better computational models for predicting the 5'-terminal exon and separating the 5' UTR from the coding region.

5' Untranslated Regions↗

PubMed2000

Isolation and characterization of a novel gene encoding a putative seven-span transmembrane protein, TM7SF3.

As part of a project involving large-scale sequencing of clones randomly selected from a human cDNA library, we isolated a novel human gene, termed TM7SF3 (transmembrane 7 superfamily member 3). Its open reading frame encodes a 570 amino acid protein containing seven putative transmembrane domains. The transcript of this gene was expressed in all human tissues examined, but most abundantlyin kidney. Immunocytochemical analysis demonstrated subcellular localization of TM7SF3 protein at the plasma membrane. We determined the chromosome location of TM7SF3 as 12q11.2-->q12 by a combination of fluorescence in situ hybridization and radiation hybrid mapping.

Amino Acid Motifs↗

PubMed2000

Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins.

We have isolated a gene, termed CORO1C (human coronin-like actin-binding protein 1C), that encodes a new member of the coronin-like family of proteins. The cDNA consists of 3,857 nucleotides, with an open reading frame of 1,422 bp encoding a 474 amino acid protein. The deduced amino acid sequence shared 65% identity with p57 (human coronin-like actin-binding protein), as well as 46% identity with coronin, a protein first isolated from the slime mold Dictyostelium discoideum. Computer analysis predicted that the product of the CORO1C gene would contain five WD repeats in its N-terminal region and a coiled-coil motif in its C-terminal region, both of which are conserved among coronin-like proteins. CORO1C was ubiquitously expressed in all human tissues examined, in contrast to other known coronin-like molecules, each of which is expressed in a tissue-specific manner. Immunocytochemical staining demonstrated that CORO1C was co-localized with F-actin; therefore, the gene product is likely to be important in cytokinesis, motility, and signal transduction, as are the other members of this molecular family. We assigned this novel gene to chromosome 12q24.1 by fluorescence in situ hybridization.

Actins↗

PubMed2000

Contribution of cyanide-insensitive respiratory pathway, catalyzed by the alternative oxidase, to citric acid production in Aspergillus niger.

In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX). In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 microM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A. niger.

Aspergillus niger↗

PubMed2000

Identification of canine alpha-lactalbumin.

The nucleotide sequence of canine alpha-lactalbumin cDNA from canine mammary tissue was determined by polymerase chain reaction with degenerate primers. A 742 base pairs nucleotide sequence cloned was similar to the size of mRNA in Northern blot analysis. The cDNA encodes 142 amino acid residues containing the conserved sequence motif of alpha-lactalbumin, demonstrating the highest homology with pig (73% identity-82% similarity) among the known amino acid sequences of alpha-lactalbumin. The canine cDNA also showed 71% identity-78% similarity with human, 58-73% with mouse, 60-74% with rat, 67-77% with goat, 66-77% with cattle, and 67-76% with sheep, respectively.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and phylogenetic analysis of canine beta-casein.

A canine beta-casein cDNA was isolated from mammary tissue by polymerase chain reaction (PCR) using degenerate primers. It encodes 250 amino acids protein containing the conserved sequence motif of beta-casein. It showed the highest homology with snow-leopard (Uncia uncia (55-62% identity). It also showed 44-53% identity with human, 33-42%, identity with mouse, 29-37%, identity with rat, 43-53% identity with rabbit, 41-48% identity with pig, 44-51% identity with cattle and 44-50% identity with sheep. A 1.2-kb mRNA was detected in mammary tissue by Northern blot analysis. Phylogenetic analysis revealed that canine beta-casein formed a branch with lesser panda and snow leopard, which were grouped into carnivore.

Amino Acid Sequence↗

PubMed1999

Cloning and expression profile of mouse and human genes, Rnf11/RNF11, encoding a novel RING-H2 finger protein.

The RING finger (C3HC4-type zinc finger) is a variant zinc finger motif presents in a new family of proteins. A new member of the RING finger family was identified and its cDNA structures were determined in human and mouse. The predicted protein consisting of a 144 amino acid residues is very conservative between the two species and contains a canonical RING-H2 finger motif (C3H2C2) at the carboxyl-terminal region. The genes were designated as RNF11/Rnf11 for RING finger protein 11. A single 2.4-kb transcript of mouse Rnf11 was ubiquitously expressed in various fetal and adult mouse tissues by the Northern blot analysis. The human RNF11 gene was mapped on chromosome 1p31-p32 region, where frequent alterations have been observed in T-cell acute lymphoblastic leukemia.

Amino Acid Sequence↗

PubMed1999

Identification of a novel protocadherin gene (PCDH11) on the human XY homology region in Xq21.3.

Protocadherins (Pcdhs) are members of the rapidly growing cadherin superfamily and are thought to be involved in cell-cell recognition in the central nervous system. Using human BH-Pcdh cDNA, we retrieved a homologous gene from the database. The new gene (Pcdh-X, HGMW-approved symbol PCDH11) was present on a genomic clone of human chromosome X (clone bWXD306), between two sequence tagged sites, sWXD1362 and 221. Pcdh-X therefore maps to the XY homology region in Xq21.3. The open reading frame consists of 1021 amino acids (aa) including seven cadherin repeats (EC1-7) in the extracellular domain. The Pcdh-X gene consists of at least three exons; the first exon encodes the 5'-untranslated region, EC1, and half of EC2, the second exon encodes the remainder of the Pcdh-Xa, and the third exon encodes the cytoplasmic tail of Pcdh-Xb and its 3'-untranslated region. The second exon has an alternative splice site that is used to produce two isoforms with different cytoplasmic tails of 10 (Pcdh-Xa) or 14 amino acids (Pcdh-Xb). Northern blot analysis revealed an approximately 6.0-kb transcript expressed in human and mouse fetal brain.

Amino Acid Sequence↗