Biomedical subjects

Takao Isogai

Publications and source records attributed to Takao Isogai.

11 recordsLinked to original sources

PubMed2006

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.

Arteries↗

PubMed2006

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56,419 completely sequenced and manually annotated full-length cDNAs.

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56,419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37,670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants.

Alternative Splicing↗

PubMed2006

A novel estrogen receptor-related protein gamma splice variant lacking a DNA binding domain exon modulates transcriptional activity of a moderate range of nuclear receptors.

A novel estrogen receptor-related protein (ERR) gamma splice variant cDNA (ERRgamma3) was found in human full-length cDNA libraries. ERRgamma3 cDNA consists of 3362 base pairs and has an open reading frame of 1188bp. The predicted peptide sequence of ERRgamma3 differs from both ERRgamma1 and ERRgamma2 in missing 39 amino acid residues corresponding to the second zinc finger motif of the DNA binding domain (DBD). ERRgamma3 gene consists of 8 exons including three unique 5'-terminal exons and lacks the exon encoding the second zinc finger motif. The expression of ERRgamma3 was confined to adipocytes and prostate while that of ERRgamma2 was fairly widespread. The ERRgamma3 product was shown by transactivation assay to have no ability to activate ERE-controlled transcription. However, ERRgamma3 has an ability to modulate the transcriptional activity of other nuclear hormone receptors. ERRgamma3 augmented the ligand-dependent transcriptional activities of ER (estrogen receptor) alpha, ERbeta, and thyroid receptor (TR) alpha by 1.3-, 4-, and 2.1-fold whereas it inhibited fully the activity of glucocorticoid receptor (GR). However, ERRgamma3 had no effect on Vitamin D3 receptor, retinoic acid receptor alpha or peroxisome proliferator activated receptor alpha, beta, and gamma. These findings will help to elucidate the physiological role of the ERRgamma subfamily.

Alternative Splicing↗

PubMed2005

Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

Base Sequence↗

PubMed2005

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose.

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) >> D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).

Amino Acid Sequence↗

PubMed2005

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

5' Flanking Region↗

PubMed2004

Molecular cloning and characterization of two novel human RXR alpha splice variants.

Two novel cDNAs encoding RXR alpha splice variants (RXR alpha 2 and RXR alpha 3) were identified among human full-length cDNA libraries. RXR alpha 2 and RXR alpha 3 cDNAs possess open reading frames, leading to production of proteins lacking the N-terminal 27 and 97 amino acid residues of the RXR alpha 1 product, respectively. RXR alpha 2 and RXR alpha 3 genes have respective 5'-terminal exons. RXR alpha 3 is expressed in brain, spleen and prostate whereas the expression of RXR alpha 2 was below the detectable level. Both RXR alpha 2 and RXR alpha 3 showed a level of transcriptional activity and a dose response curve against the agonist LG100268 similar to RXR alpha 1 in reporter assay for the RXR alpha homodimer or that for the heterodimer with PPAR gamma 2. However, clear differences were observed among the splice variants when dose response curves were compared by the assay in the presence of coactivators such as SRC-1 and PGC-1. These results suggest specific physiological roles of two novel human RXR alpha splice variants.

Alternative Splicing↗

PubMed2003

Salusins: newly identified bioactive peptides with hemodynamic and mitogenic activities.

The discovery of endogenous bioactive peptides has typically required a lengthy identification process. Computer-assisted analysis of cDNA and genomic DNA sequence information can markedly shorten the process. A bioinformatic analysis of full-length, enriched human cDNA libraries searching for previously unidentified bioactive peptides resulted in the identification and characterization of two related peptides of 28 and 20 amino acids, which we designated salusin-alpha and salusin-beta. Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. Intravenous administration of salusin-alpha or salusin-beta to rats causes rapid, profound hypotension and bradycardia. Salusins increase intracellular Ca2+, upregulate a variety of genes and induce cell mitogenesis. Salusin-beta stimulates the release of arginine-vasopressin from rat pituitary. Expression of TOR2A mRNA and its splicing into preprosalusin are ubiquitous, and immunoreactive salusin-alpha and salusin-beta are detected in many human tissues, plasma and urine, suggesting that salusins are endocrine and/or paracrine factors.

Adenosine Triphosphatases↗

PubMed2003

Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis.

Characterization of dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes is of biological and clinical interest. We isolated DECs and bone marrow stromal cells (BMSCs), H4-1 and H3-4, and demonstrated that the cells started to produce extracellular matrices, such as type II collagen and aggrecan, at an early stage of chondrosphere formation. Furthermore, cDNA sequencing of cDNA libraries constricted by the oligocapping method was performed to analyze difference in mRNA expression profiling between DECs and marrow stromal cells. Upon redifferentiation of DECs, cartilage-related extracellular matrix genes, such as those encoding leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and chitinase 3-like 1 (cartilage glycoprotein-39), were highly expressed. Growth factors such as FGF7 and CTGF were detected at a high frequency in the growth stage of monolayer stromal cultures. By combining the expression profile and flow cytometry, we demonstrated that isolated stromal cells, defined by CD34(-), c-kit(-), and CD140alpha(- or low), have chondrogenic potential. The newly established human mesenchymal cells with expression profiling provide a powerful model for a study of chondrogenic differentiation and further understanding of cartilage regeneration in the means of redifferentiated DECs and BMSCs.

Bone Marrow Cells↗

PubMed2002

cDNA macroarray analysis of gene expression in synoviocytes stimulated with TNFalpha.

Gene expression of synoviocytes stimulated with tumor necrosis factor-alpha (TNFalpha) was studied by macroarray analysis to elucidate the cellular response and identify new biological functions of known and unknown genes. 10035 cDNA clones were used to make cDNA macroarrays of representative genes. Synoviocytes expressed large amounts of fibronectin and collagen mRNA. Statistical analysis of the macroarray data revealed 26 genes, including six new genes, which underwent significant alteration of gene expression in response to TNFalpha stimulation. These findings suggest that the synoviocyte response to TNFalpha stimulation forms the basis of development of various aspects of the pathophysiology of rheumatoid arthritis.

Amino Acid Sequence↗

PubMed2002

Database and analysis system for cDNA clones obtained from full-length enriched cDNA libraries.

We have developed an efficient sequence-analysis system and a database system for clones obtained from full-length enriched cDNA libraries made by using the oligo-capping method. We developed a semi-automatic analysis system for 5'- and 3'-end sequences. It pre-processes raw sequences (vector cut and accurate-sequence region extraction), clusters the sequences, searches for similarities through public databases, annotates completeness of clones and analyzes the ORFs in the sequences. Newly developed or improved programs are used in each step. A new program, ESTiMateFull is used to evaluate and to predict the sequence-fullness based on comparisons with mRNA and EST sequences, respectively. The ATGpr program is used to predict sequence-fullness based on statistical information. The combination of full-length enriched cDNA clones and ATGpr fullness prediction resulted in 70% accuracy in the specificity and the sensitivity of the fullness predictions. For the ORFs predicted by the ATGpr, the signal peptides are predicted and a motif search is performed by our new system. We also developed a program that assembles our sequences with dbEST sequences and developed a system to retrieve clones by the characteristics of the ORFs. As keywords, combination of various results of the analyses can be used for retrieval. And various results such as ORF features and database search results can be shown on the same screen by multiple displays. Full-length clones having interesting functions can thus be retrieved efficiently by using this system.

Amino Acid Sequence↗