Biomedical subjects

S Sugano

Publications and source records attributed to S Sugano.

At least 19 recordsLinked to original sources

PubMed2006

Stability of canine distemper virus (CDV) after 20 passages in Vero-DST cells expressing the receptor protein for CDV.

Isolates 007Lm, S124C and Ac96I and a Vero cell-adapted Onderstepoort strain of canine distemper viruses (CDV) were examined for stability after passages in Vero cells expressing the canine signaling lymphocyte activation molecule (dogSLAM, the intrinsic receptor to CDV). These viruses passage once in Vero cells expressing dogSLAM (Vero-DST) cells (original) and after 20 passages (20p) were compared by using sequence analyses and growth characteristics. All four strains of 20p grew well and were slightly better than their originals. The 20p viruses developed a cytopathic effect slightly lower than the original strains. A few changes in amino acids in the H gene were between the 20p and the original viruses, but the sites of changes were not specific. Fragments of P, M and L genes of all strains showed no nucleotide changes after the passages. These results showed that: (1) passages of CDVs in Vero-DST cells induced amino acid changes only in the H gene, not in the P, M and L genes, unlike in a previous study with Vero cells; (2) passages did not markedly affect the growth characteristics of every viral strain. These results indicate that Vero cells expressing canine SLAM allow the isolation and passaging of CDV without major changes in viral genes.

Amino Acid Sequence↗

PubMed2006

Comparative analyses of canine distemper viral isolates from clinical cases of canine distemper in vaccinated dogs.

Sequence and phylogenetic analyses of three isolates of canine distemper virus (CDV) isolated from three dogs with a vaccination history were compared with the same analyses of vaccine virus isolated from a vaccine used for dogs. The three dogs showed clinical signs of a recent major type of CD in Japan, including oculonasal discharge and diarrhea, and pathological findings including non-suppurative encephalitis, pneumonia, mild gastroenteritis and lymphoid depletion. Inclusion bodies were in the stomach without inflammation and encephalitis was without clinical signs. One of the highest titers of CDV in different organs of the three dogs was commonly systemic lymphatic organs, including the spleen, lymph nodes and tonsils. New isolates of CDV joined to the clades of the Asia 1 group that is far from the vaccine group. These results surely indicate that wild strains of CDV from dogs with a vaccination history were not reversed vaccine virus, and that the dogs showed characteristics of recent CD in Japan.

Amino Acid Sequence↗

PubMed2006

Restoration of transforming growth factor-beta type II receptor reduces tumorigenicity in the human adrenocortical carcinoma SW-13 cell line.

Transforming growth factor-beta (TGF-beta) is a potent growth suppressor. Acquisition of TGF-beta resistance has been reported in many tumors, and has been associated with reduced TGF-beta receptor expression. In this study, we examined TGF-beta 1, TGF-beta type I receptor (TbetaRI) and TGF-beta type II receptor (TbetaRII) expression in SW-13 adrenocortical carcinoma cells by Northern and Western blot analysis. SW-13 cells did not express TbetaRII mRNA or protein. We have investigated the role of TbetaRII in modulating tumorigenic potential using stably transfected SW-13 cells with TbetaRII expression plasmid. TbetaRII-positive SW-13 cell growth was inhibited by exogenous human TGF-beta1 (hTGF-beta1) in a dose-dependent manner. In contrast, SW-13 cells and control clones transfected with empty vector remained hTGF-beta1-insensitive. Xenograft examination in athymic nude mice demonstrated that TbetaRII-positive SW-13 cells reduced tumor-forming activity. Reconstructing the TbetaRII can lead to reversion of the malignant phenotype of TbetaRII-negative human adrenocortical carcinoma, which contains SW-13 cells. Reduced TbetaRII expression may play a critical role in determining the malignant phenotype of human adrenocortical carcinoma.

Adrenal Cortex Neoplasms↗

PubMed2005

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours.

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.

14-3-3 Proteins↗

PubMed2005

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

ATP-Binding Cassette Transporters↗

PubMed2005

Growth profiles of recent canine distemper isolates on Vero cells expressing canine signalling lymphocyte activation molecule (SLAM).

Fresh samples of lymph node, lung and cerebrum taken post mortem from dogs no. 1, 2 and 3 yielded canine distemper virus (CDV) strains 007 Lm, 009 L and 011 C, respectively. These were titrated on Vero cells stably expressing canine signalling lymphocyte activation molecule (SLAM; Vero-DST cells). Growth curves of the three strains were produced by titration of the released virus and cell-associated virus at various timepoints. All three isolates, especially 007 Lm, grew well on Vero-DST cells. The titres of cell-associated virus of two strains (009 L and 011 C) were clearly lower than those of virus released into the culture supernate. The results indicate that Vero-DST cells are not only useful for primary isolation but also efficient for titrating virus from fresh tissues and for the study of growth profiles of recent CDV isolates.

Animals↗

PubMed2005

A steroidogenic cell line with differentiation potential from mouse granulosa cells, transfected with Ad4BP and SV40 large T antigen genes.

Several steroidogenic cell lines of granulosa cells (GC) have been used to elucidate differentiation mechanisms of GC during folliculogenesis. These cell lines, however, are of limited usefulness since they have lost some of their differentiation potential. The transcription factor adrenal-4 binding protein (Ad4BP), also known as steroidogenic factor-1 or NR5A1, is essential for the expression of all P-450 steroidogenic enzymes. By transfection with the Ad4BP gene together with SV40 DNA, we have generated several steroidogenic cell lines. One selective clone, named 4B2, retained its steroidogenic potential and was therefore analyzed in depth. This cell line responded to 8-Br-cAMP by displaying differentiation characteristics similar to those occurring in the differentiation process of primary cultured GC, including enhanced progesterone secretion, a cell shape change from a fibroblastic to epithelioid conformation, elongated mitochondria, increased gap junction formation and inhibition of cell proliferation. Prostaglandin E2 (PGE2), an intraovarian regulator of GC, stimulated cAMP production, and this eicosanoid, like 8-Br-cAMP, induced differentiation properties with the exception of cell conformation in 4B2 cells. These results suggest that expression of Ad4BP may provide the basis for a repertoire of cAMP-sensitive differentiation properties, including morphological alterations and growth inhibition. Thus, the 4B2 cell line may serve as a tool for elucidation of differentiation mechanisms that are under the control of Ad4BP.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2001

Comparison of sequences of cDNA clones obtained from oligo-capping cDNA libraries with those from unigene.

We compared in detail the characteristics of the sequences of the cDNA clones obtained by the oligo-capping method (oligo-capping clones) with that of the sequences in the UniGene database. To compare the completeness of the sequences, three new variables, "fullness-proportion of clones" (the ratio of complete clones to total clones in a library), "fullness-proportion of genes" (the ratio of complete genes to total genes in a library), and "fullness-proportion of database" (the ratio of complete genes to total genes in a database sampled from a library), were defined. The fullness-proportion of clones of oligo-capping clones was 57.3%, 2.2 times larger than that of UniGene (25.9%). The fullness-proportion of genes of oligo-capping clones was 41.8%, 2.4 times larger than that of UniGene (17.8%). When gene length was restricted to > or = 1.5 kb, the fullness-proportion of genes of oligo-capping clones was four times larger than that of UniGene. The fullness-proportion of database of oligo-capping clones was approximately the same as that of UniGene. By simulating the clone redundancy, this coincidence was found to be due to the large redundancy of the UniGene database. Consequently, the cDNA sequence database of oligo-capping clones enabled high throughput selection of full-length cDNA clones.

Algorithms↗

PubMed2001

Identification of sonic hedgehog-responsive genes using cDNA microarray.

Sonic hedgehog (Shh) is a secreted signaling protein that plays important roles in a variety of developmental processes and also in pathogenesis of some human cancers and congenital diseases. Molecules that function downstream of Shh, however, still remain elusive. Here we searched for Shh-responsive genes by using an in-house cDNA microarray. Two genes were newly identified to be Shh responsive in neuroepithelial cell line MNS-70: the metal-binding protein Ceruloplasmin (Cp) and the serine protease inhibitor inter-alpha-trypsine inhibitor heavy chain H3 (ITIH3). In MNS-70 cells, expression of ITIH3 was regulated by Gli zinc-finger transcription factors downstream of Shh, whereas Cp appeared to be regulated by Gli-independent pathways. Cp mRNA was detected in the developing mouse brain, where its expression domain was closely adjacent to that of Shh. These results demonstrate that microarray technology provides a useful tool for studying expression of developmentally regulated genes.

Animals↗

PubMed2001

Molecular characterization of NSD1, a human homologue of the mouse Nsd1 gene.

NR-binding SET-domain-containing protein (NSD1) is a mouse nuclear protein containing su(var)3-9, enhancer-of-zeste, trithorax (SET), proline-tryptophan-tryptophan-proline (PWWP) and plant homeodomain protein (PHD)-finger domains (Huang et al., EMBO J. 17 (1998) 3398). This protein also has two other distinct nuclear receptor (NR)-interaction domains, called NID(-L) and NID(+L), and acts as both a NR corepressor and a coactivator by interacting directly with the ligand-binding domain of several NRs. Thus, NSD1 is a bifunctional, transcriptional, intermediary factor. We isolated the human homologue (NSD1) of the mouse NSD1 gene (Nsd1), mapped it to human chromosome 5q35, and characterized its genomic structure. NSD1 consists of at least 23 exons. Its cDNA is 8552 bp long, has an 8088 bp open reading frame, contains at least six functional domains (SET, PWWP-I, PWWP-II, PHD-I, PHD-II, and PHD-III) and ten putative nuclear localization signals, and encodes 2696 amino acids. NSD1 shows 86% identity with the mouse Nsd1 at the nucleotide level, and 83% at the amino acid level. NSD1 is expressed in the fetal/adult brain, kidney, skeletal muscle, spleen, and the thymus, and faintly in the lung. Two different transcripts (9.0 and 10.0 kb) were consistently observed in various fetal and adult tissues examined. These findings favor the character of NSD1 as a nucleus-localized, basic transcriptional factor and also a bifunctional transcriptional regulator, such as that of the mouse Nsd1. It remains to be investigated whether mutations of NSD1 lead to a specific phenotype in man.

Amino Acid Sequence↗

PubMed2001

Purification of cytochromes P-450(scc) and P-450(17 alpha) by steroid-binding affinity column chromatography.

The preparation, testing and use of a variety of cholesterol-, deoxycorticosterone (DOC)- and pregnenolone-binding 1,6-diaminohexyl (EAH)-Sepharose 4B supports for affinity column chromatography of cytochromes P-450(scc) and P-450(17 alpha) from bovine adrenal and pig testis are described. EAH-Sepharose 4B has free amino groups at the end of a 10-atom spacer arm. Hydroxyl groups of cholesterol (3 beta), deoxycorticosterone (21 beta) and pregnenolone (3 beta) are linked to succinic anhydride in pyridine through an ester linkage. These coupling ligands of hemisuccinate were synthesized by a general procedure. Free amino groups of EAH-Sepharose 4B were used to couple ligands, containing carboxyl groups, by the carbodiimide coupling method. Both the purified cytochromes P-450(scc) and P-450(17 alpha) were found to be homogeneous and estimated to have a molecular weight of 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectra with peaks at 450 and 448 nm exhibit the absorption spectra of typical cytochromes P-450(scc) and P-450(17 alpha), respectively. Cytochromes P-450(scc) and P-450(17 alpha) were determined to have isoelectric points of 8.0 and 6.5 in isoelectric focusing on a pH gradient gel. Cytochrome P-450s can be purified between 425- and 1000-fold from the crude extracts.

Adrenal Glands↗

PubMed2001

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes.

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.

Animals↗

PubMed2001

Structural analyses of the UTF1 gene encoding a transcriptional coactivator expressed in pluripotent embryonic stem cells.

The UTF1 is a transcriptional coactivator expressed mainly in pluripotent embryonic stem cells. Here, we have isolated a genomic DNA fragment carrying the UTF1 gene and found that the gene contains two exons interrupted by a short intron. The gene possesses four GC boxes, but no TATA box in the 5'-flanking region. This is reminiscent of a housekeeping gene type promoter and the functional relevance of these motifs is confirmed by the transient transfection analyses. As to the gene product, our analyses have led to the identification of two different species. One of them corresponds to the full-length protein, while the other is produced by utilizing the second methionine codon for the translation initiation. The oligo-capping analyses reveal multiple transcription start sites. Interestingly, some of them are localized downstream of the first methionine codon, indicating that such transcripts produce a protein starting from the second methionine codon. Chromosomal mapping analyses locate the gene at 7F5, the syntenic region of the human chromosome (10q26) where the human UTF1 gene is located.

Amino Acid Sequence↗

PubMed2001

CREB-H: a novel mammalian transcription factor belonging to the CREB/ATF family and functioning via the box-B element with a liver-specific expression.

The expression of liver-specific genes is regulated by unequivocally allocated transcription factors via proper responsible elements within their promoters. We identified a novel transcription factor, CREB-H, and found that its expression was restricted in the liver among 16 human tissues tested. A region of CREB-H exhibited significant homology to the basic leucine zipper (b-Zip) domain of members of the CREB/ATF family: mammalian LZIP and Drosophila BBF-2 that binds to box-B, a Drosophila enhancer modulating the fat-body-specific gene expression. CREB-H contained a hydrophobic region representing a putative transmembrane domain, like LZIP. Constructing a variety of CREB-H fusion proteins with the GAL4 DNA-binding domain disclosed that CREB-H functioned as a transcriptional activator and its N-terminal 149 amino acids accounted for the activation ability. Gel mobility sift assays revealed that CREB-H did not bind to the C/EBP, AP-1 and NF-kappaB elements but specifically bound to CRE and the box-B element. Luciferase reporter assays demonstrated that like BBF-2, CREB-H activated transcription via the box-B element and that a deletion of the putative transmembrane domain increased the activation of reporter expression significantly. Furthermore, a fusion protein of GFP and full-length CREB-H was localized in reticular structures surrounding the nucleus, whereas a fusion protein of GFP and a deletion mutant lacking the putative transmembrane domain was mainly in the nucleus. These findings suggest that CREB-H plays an important role in transcriptional regulation of genes specifically expressed in the liver, and that the putative transmembrane domain may be associated with modulation of its function as the transcriptional activator.

Activating Transcription Factors↗

PubMed2001

The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast.

The Schizosaccharomyces pombe dhp1(+) gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5'-->3' exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)(+) RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1(+) is required for proper chromosome segregation as well as for poly(A)(+) RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1(+). We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

Cell Division↗

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

FULL-malaria: a database for a full-length enriched cDNA library from human malaria parasite, Plasmodium falciparum.

FULL-malaria is a database for a full-length-enriched cDNA library from the human malaria parasite Plasmodium falciparum (http://133.11. 149.55/). Because of its medical importance, this organism is the first target for genome sequencing of a eukaryotic pathogen; the sequences of two of its 14 chromosomes have already been determined. However, for the full exploitation of this rapidly accumulating information, correct identification of the genes and study of their expression are essential. Using the oligo-capping method, we have produced a full-length-enriched cDNA library from erythrocytic stage parasites and performed one-pass reading. The database consists of nucleotide sequences of 2490 random clones that include 390 (16%) known malaria genes according to BLASTN analysis of the nr-nt database in GenBank; these represent 98 genes, and the clones for 48 of these genes contain the complete protein-coding sequence (49%). On the other hand, comparisons with the complete chromosome 2 sequence revealed that 35 of 210 predicted genes are expressed, and in addition led to detection of three new gene candidates that were not previously known. In total, 19 of these 38 clones (50%) were full-length. From these observations, it is expected that the database contains approximately 1000 genes, including 500 full-length clones. It should be an invaluable resource for the development of vaccines and novel drugs.

Animals↗

PubMed2001

Diversification of the microtubule system in the early stage of eukaryote evolution: elongation factor 1 alpha and alpha-tubulin protein phylogeny of termite symbiotic oxymonad and hypermastigote protists.

The symbiotic protists of the lower termite have been regarded as a model of early-branched eukaryotes because of their simple cellular systems and morphological features. However, cultivation of these symbiotic protists is very difficult. For this reason, these interesting protists have not been well characterized in terms of their molecular biology. In research on these organisms which have not yet been cultivated, we developed a method for retrieving specific genes from a small number of cells, through micromanipulation without axenic cultivation, and we obtained EF-1 alpha and alpha-tubulin genes from members of the Hypermastigida--the parabasalid protist Trichonympha agilis and the oxymonad protists Pyrsonympha grandis and Dinenympha exilis--from the termite Reticulitermes speratus gut community. Results of phylogenetic analysis of the amino acid sequences of both proteins, EF-1 alpha and alpha-tubulin, indicate that the hypermastigid, parabasalid, and oxymonad protists do not share a close common ancestor. In addition, although the EF-1 alpha phylogeny indicates that these two groups of protists branched at an early stage of eukaryotic evolution, the alpha-tubulin phylogeny indicates that these protists can be assigned to two diversified clades. As shown in a recent investigation of alpha-tubulin phylogeny, eukaryotic organisms can be divided into three classes: an animal--parabasalids clade, a plant--protists clade, and the diplomonads. In this study, we show that parabasalids, including hypermastigids, can be classified as belonging to the animal--parabasalids clade and the early-branching eukaryote oxymonads can be classified as belonging to the plant--protists clade. Our findings suggest that these protists have a cellular microtubule system that has diverged considerably, and it seems that such divergence of the microtubule system occurred in the earliest stage of eukaryotic evolution.

Animals↗