Biomedical subjects

Susumu Katsuma

Publications and source records attributed to Susumu Katsuma.

At least 19 recordsLinked to original sources

PubMed2006

Glucocorticoid-induced alternative promoter usage for a novel 5' variant of granzyme A.

Glucocorticoids exert diverse physiological functions through transcriptional regulation of genes including granzyme A (GZMA). GZMA is one of the apoptotic effectors localized in cytotoxic T lymphocytes and is considered to mediate glucocorticoid-induced apoptosis of human leukemia 697 cells. In the present study, we identified a novel 5' variant transcript of GZMA in dexamethasone (DEX)-treated 697 cells. We designated this novel transcript as GZMAbeta. The transcription of GZMAbeta starts at 290 bp downstream of the first intronic glucocorticoid response element (GRE). Chromatin immunoprecipitation assay showed that glucocorticoid receptor (GR) binds to the intronic GRE in a DEX-dependent manner. Luciferase assay and RT-PCR also showed that DEX induces GZMAbeta transcription mediated by GR binding to the intronic GRE. Our results show that there exist at least two transcripts in human GZMA, whose expression is differentially regulated by glucocorticoid.

5' Untranslated Regions↗

PubMed2006

Mutational analysis of active site residues of chitinase from Bombyx mori nucleopolyhedrovirus.

Infection of Bombyx mori larvae with B. mori nucleopolyhedrovirus (BmNPV) results in liquefaction of the host. This process is attributed to the synergistic action of two virus-encoded genes, chitinase (v-chiA) and cathepsin (v-cath). Previous studies have suggested that Autographa californica nucleopolyhedrovirus (AcMNPV) CATH cannot be processed within infected cells in the absence of AcMNPV CHIA. To investigate the interactions between V-CHIA and V-CATH, we generated a recombinant BmNPV (103ChiAmut) in which the residues of the active site of BmNPV chiA were mutated (D302NE306Q) and the gene was driven by its own promoter at the native locus. Mutation at the active site of BmNPV CHIA resulted in complete loss of chitinolytic activity. Bombyx mori larvae infected with 103ChiAmut survived longer than larvae infected with wild-type BmNPV and did not undergo terminal liquefaction after death. Cysteine protease activity and Western blot analysis showed that, in cells infected with v-chiA-deleted BmNPV (ChiAD), BmNPV CATH was not processed properly and was accumulated as a detergent-insoluble form, suggesting that BmNPV CHIA plays a crucial role in V-CATH processing. In cells infected with 103ChiAmut, BmNPV CATH formed insoluble aggregates, suggesting that active site-mutated BmNPV CHIA loses its additional role as a molecular chaperon during V-CATH processing.

Amino Acid Substitution↗

PubMed2006

N-linked glycans of Bombyx mori nucleopolyhedrovirus fibroblast growth factor are crucial for its secretion.

Bombyx mori nucleopolyhedrovirus (BmNPV) fibroblast growth factor (BmFGF) is a glycosylated protein that is efficiently secreted into the medium. Here, we constructed mutant NPVs expressing His-tagged wild-type (wt) or mutant BmFGFs and showed that the two residues, asparagine 44 and 171, are the glycosylation sites of BmFGF. Also, removal of N-linked glycans from BmFGF resulted in almost complete inhibition of the secretion into the medium, suggesting that N-linked glycans of BmFGF are required for its secretion. These residues are not conserved in closely related Autographa californica NPV (AcMNPV)-encoded vFGF (AcFGF). Western blot analysis suggested that AcFGF is not glycosylated and is poorly secreted. A mutant AcFGF possessing two N-linked glycosylation sites was secreted into the medium more abundantly than that which occurred for wt AcFGF. This is the first direct evidence showing the role of N-linked glycans in the secretion process of a baculovirus protein.

Amino Acid Sequence↗

PubMed2006

In vivo and in vitro analyses of a Bombyx mori nucleopolyhedrovirus mutant lacking functional vfgf.

All lepidopteran baculovirus genomes sequenced to date encode a viral fibroblast growth factor homolog (vfgf), suggesting that vfgf may play an important role in the infection cycle of lepidopteran baculoviruses. Here, we describe the characterization of a Bombyx mori nucleopolyhedrovirus (BmNPV) mutant lacking functional vfgf. We constructed a vfgf deletion mutant (BmFGFD) and characterized it in BmN cells and B. mori larvae. We observed that budded virus (BV) production was reduced in BmFGFD-infected BmN cells and B. mori larvae. The larval bioassays also revealed that deletion of vfgf did not reduce the infectivity; however, the mutant virus did take 20 h longer to kill B. mori larvae than wild-type BmNPV, when tested either by BV injection or by polyhedrin-inclusion body ingestion. These results suggest that BmNPV vfgf is involved in efficient virus production in BmN cells and B. mori larvae.

Animals↗

PubMed2006

Comparative studies of Bombyx mori nucleopolyhedrovirus chitinase and its host ortholog, BmChi-h.

Baculovirus-encoded chitinases (V-CHIAs) were first proposed to be acquired from a bacterium via horizontal gene transfer. However, we have recently reported that lepidopteran hosts also encode v-chiA orthologs. Here we describe comparative studies of Bombyx mori nucleopolyhedrovirus (BmNPV) chitinase and its host ortholog, BmChi-h. We constructed recombinant BmNPVs in which native and modified forms of BmChi-h were driven under the polyhedrin promoter and the authentic v-chiA was deleted. Western blot analysis indicated that BmCHI-h was rapidly secreted from virus-infected BmN cells whereas BmNPV CHIA was localized within the virus-infected cells; probably because of the presence of a C-terminal endoplasmic reticulum retention motif on BmNPV CHIA. Enzymological studies showed that BmNPV CHIA was able to retain much higher chitinolytic activity under alkaline conditions. For B. mori larvae infected with v-chiA-deleted BmNPV, the terminal liquefaction of dead larvae and the activation of baculovirus-encoded cysteine protease were not observed, and the introduction of BmChi-h did not rescue these defects. Our findings show that BmNPV chiA possesses unique features that are not shared by host orthologs, which may reflect functional specialization of baculovirus chitinases.

Animals↗

PubMed2006

Developmental aberrations of liver gene expression in bovine fetuses derived from somatic cell nuclear transplantation.

Cloning by somatic cell nuclear transfer (NT) has been accomplished. However, the process itself is inefficient since most clones die before birth and survivors often display various anomalies. In an effort to determine global expression profiles of developmentally regulated liver genes in NT bovine fetuses, we employed a custom-made bovine liver complementary DNA (cDNA) microarray. The NT fetuses in early pregnancy were derived from cumulus cells as the nuclear donor cells. Normal fetuses were derived from in vitro fertilization (IVF) and artificial insemination (AI). Gene expression levels in NT, IVF, and AI fetal livers were obtained by comparing individual fetal liver samples with that of adult liver of nonpregnant cycling cows. Statistical analyses of the expression data showed widespread dysregulation of developmentally important genes in the three NT fetuses examined. It was found that the number of dysregulated genes was within a range of 3.5-7.7% of the tested genes in the NT fetal livers. The analyses revealed that one NT fetus was markedly different in liver gene expression profile from the other two NT fetal livers in which the expression profiles were highly correlated. Thus, our findings demonstrate that widespread dysregulation of liver genes occurs in the developing liver of NT bovine fetuses. It is possible that inappropriate genomic reprogramming after NT is a key factor associated with abnormal gene expressions in the livers of NT fetuses, whereas distinct expression patterns between the fellow cloned fetuses likely have resulted from variable epigenetic status of the donor nuclei.

Animals↗

PubMed2006

Lepidopteran ortholog of Drosophila breathless is a receptor for the baculovirus fibroblast growth factor.

The Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a gene homologous to the mammalian fibroblast growth factor (FGF) family. We report the cloning of B. mori and Spodoptera frugiperda orthologous genes (Bmbtl and Sfbtl, respectively) of Drosophila melanogaster breathless (btl) encoding a receptor for Branchless/FGF and show that these genes encode the receptor for a baculovirus-encoded FGF (vFGF). Sequence analysis showed that BmBtl is composed of 856 amino acid residues, which potentially encodes a 97.3-kDa polypeptide and shares structural features and sequence similarities with the FGF receptor family. Reverse transcription-PCR experiments showed that Bmbtl was abundantly expressed in the trachea and midgut in B. mori larvae, with moderate expression observed in the hemocytes and the B. mori cultured cell line BmN. We generated Sf-9 cells that stably expressed His-tagged BmBtl. Western blot analysis revealed that BmBtl was an approximately 110-kDa protein. Immunoprecipitation experiments showed that BmNPV vFGF markedly phosphorylated BmBtl in Sf-9 cells. In addition, we found that BmBtl overexpression enhanced the migration activity for BmNPV vFGF. Furthermore, we generated Sf-9 cells in which Sfbtl was knocked down by transfection with double-strand RNA-expressing plasmids. In these cells, cell motility triggered by vFGF was markedly reduced. These results strongly suggest that the Btl orthologs, BmBtl and SfBtl, are the receptors for vFGF, which mediate vFGF-induced host cell chemotaxis.

Animals↗

PubMed2005

Inhibition of protein kinase CK2 prevents the progression of glomerulonephritis.

Glomerulonephritis (GN) is a progressive inflammation that may be caused by a variety of underlying disorders. It is the primary cause of chronic renal failure and end-stage renal disease, which require dialysis and transplantation worldwide. Immunosuppressive therapy has been used to treat GN clinically, but this treatment has had insufficient therapeutic effects. Here, we show that protein kinase CK2 is a key molecule in the progression of GN. cDNA microarray analysis identified CK2alpha, the catalytic subunit of CK2, as a GN-related, differentially expressed gene. Overexpression of CK2alpha was noted in the proliferative glomerular lesions in rat GN models and in renal biopsy specimens from lupus nephritis or IgA nephropathy patients. Administration of either antisense oligodeoxynucleotide against CK2alpha or low molecular weight CK2-specific inhibitors effectively prevented the progression of renal pathology in the rat GN models. The resolution of GN by CK2 inhibition may result from its suppression of extracellular signal-regulated kinase-mediated cell proliferation, and its suppression of inflammatory and fibrotic processes that are enhanced in GN. Our results show that CK2 plays a critical role in the progression of immunogenic renal injury, and therefore, CK2 is a potential target for GN therapy.

Analysis of Variance↗

PubMed2005

Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells.

Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-beta) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-beta does.

Animals↗

PubMed2005

Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1.

Bile acids play essential roles in the absorption of dietary lipids and in the regulation of bile acid biosynthesis. Recently, a G protein-coupled receptor, TGR5, was identified as a cell-surface bile acid receptor. In this study, we show that bile acids promote glucagon-like peptide-1 (GLP-1) secretion through TGR5 in a murine enteroendocrine cell line STC-1. In STC-1 cells, bile acids promoted GLP-1 secretion in a dose-dependent manner. As STC-1 cells express TGR5 mRNA, we examined whether bile acids induce GLP-1 secretion through TGR5. RNA interference experiments showed that reduced expression of TGR5 resulted in reduced secretion of GLP-1. Furthermore, transient transfection of STC-1 cells with an expression plasmid containing TGR5 significantly enhanced GLP-1 secretion, indicating that bile acids promote GLP-1 secretion through TGR5 in STC-1 cells. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in STC-1 cells. An adenylate cyclase inhibitor, MDL12330A, significantly suppressed bile acid-promoted GLP-1 secretion, suggesting that bile acids induce GLP-1 secretion via intracellular cAMP production in STC-1 cells.

Adenylyl Cyclase Inhibitors↗

PubMed2005

Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1.

Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.

Animals↗

PubMed2005

The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process.

The silkworm, Bombyx mori, has been recently demonstrated to contain a bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h showed extensive structural similarities with chitinases from bacteria such as Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase genes have not been found from any eukaryotes and viruses except for lepidopteran insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may be derived from a bacterial or baculovirus chitinase gene via horizontal gene transfer. In this report, we investigated the biological function of BmChi-h. Our enzymological study indicated that a chitinase encoded by BmChi-h has exo-type substrate preference, which is the same as S. marcescens chiA and v-CHIA, and different from BmChitinase, which has endo-type substrate preference. An immunohistochemical study revealed that BmChi-h localizes in the chitin-containing tissues during the molting stages, indicating that it plays a role in chitin degradation during molting. These results suggest that BmChi-h (exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that bacterial-type chitinase genes are highly conserved among lepidopteran insects, suggesting that the utilization of a bacterial-type chitinase during the molting process may be a general feature of lepidopteran insects.

Amino Acid Sequence↗

PubMed2005

Role of Chk1 and Chk2 in Ara-C-induced differentiation of human leukemia K562 cells.

Human chronic myelogenous leukemia K562 cells are relatively resistant to the anti-metabolite cytosine arabinoside (Ara-C) and, when treated with Ara-C, they differentiate into erythrocytes without undergoing apoptosis. In this study we investigated the mechanism by which Ara-C induces K562 cells to differentiate. We first observed that Ara-C-induced differentiation of these cells is completely inhibited by the radiosensitizing agent caffeine, an inhibitor of ATM and ATR protein kinases. We next found that Ara-C activates Chk1 and Chk2 in the cells, and that the activation of Chk1, but not of Chk2, was almost completely inhibited by caffeine. Proteasome-mediated degradation of Cdc25A and phosphorylation of Cdc25C were induced by Ara-C treatment, presumably due to the activation of Chk2 and Chk1, respectively. To directly observe the effects of checkpoint kinase activation in Ara-C-induced differentiation, we suppressed Chk1 or Chk2 with the Chk1-specific inhibitor Go6976, by generating cell lines stably over-expressing dominant-negative forms of Chk2, or by siRNA-mediated knock-down of the Chk1 or the Chk2 gene. The results suggest that Ara-C-induced erythroid differentiation of K562 cells depends on both Chk1 and Chk2 pathways.

Antimetabolites, Antineoplastic↗

PubMed2005

Novel macula-like virus identified in Bombyx mori cultured cells.

We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells.

Amino Acid Sequence↗

PubMed2004

Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120.

Diabetes, a disease in which the body does not produce or use insulin properly, is a serious global health problem. Gut polypeptides secreted in response to food intake, such as glucagon-like peptide-1 (GLP-1), are potent incretin hormones that enhance the glucose-dependent secretion of insulin from pancreatic beta cells. Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules in various cellular processes, including the secretion of gut incretin peptides. Here we show that a G-protein-coupled receptor, GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain FFAs. Furthermore, we show that the stimulation of GPR120 by FFAs promotes the secretion of GLP-1 in vitro and in vivo, and increases circulating insulin. Because GLP-1 is the most potent insulinotropic incretin, our results indicate that GPR120-mediated GLP-1 secretion induced by dietary FFAs is important in the treatment of diabetes.

Animals↗

PubMed2004

Expression profiling of baculovirus genes in permissive and nonpermissive cell lines.

The baculoviruses Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) are highly homologous at the genomic level, but they have essentially nonoverlapping host ranges. In order to characterize baculovirus replication in permissive and nonpermissive cell lines, the expression profiles of baculovirus-specific genes (at 2, 6, 12, 24, 36, 48 or 72 h post-infection) were examined in BmN (BmNPV-permissive) or Sf-9 (AcMNPV-permissive) cells that were inoculated with BmNPV or AcMNPV. Surprisingly, nearly all of the 154 genes of AcMNPV appeared to be expressed in both Sf-9 and BmN cells although the peak expression levels of these genes were delayed by roughly 12 h in the nonpermissive BmN cells. In addition, the expression levels of the very late AcMNPV polyhedrin and p10 genes were dramatically reduced in BmN cells, which presumably led to the inability of AcMNPV to form polyhedral inclusion bodies in BmN cells. Nearly all of the 136 genes of BmNPV appeared to be expressed in BmN cells, however, BmNPV gene expression was dramatically reduced in Sf-9 cells inoculated with BmNPV. Experiments in which BmNPV DNAs were transfected to Sf-9 cells suggested that this dramatic reduction in gene expression was not the result of poor attachment, penetration or uncoating of the BmNPV virion into Sf-9 cells. In conclusion, we established a system to monitor global gene expression patterns during baculovirus infection in permissive and nonpermissive cell lines. This system was used to identify global trends in the transcription of baculovirus genes during productive and nonproductive infection.

Animals↗

PubMed2004

Gene expression profile in experimental mesangial proliferative glomerulonephritis.

We analyzed gene expression in rat anti-Thy1 antibody-induced glomerulonephritis by using the cDNA microarray method. Ninety-seven genes that differed by more than 1.5-fold intensity in comparison with the controls were selected. Cluster analysis showed that the expression of genes associated with inflammation reached maximum levels at 24 h, while genes involved in the development of fibrosis increased at 7 days after injection. Microarray analysis of animal disease models may be a powerful approach for understanding the gene expression programs that underlie these disorders.

Animals↗

PubMed2004

Pregnancy-associated changes in genome-wide gene expression profiles in the liver of cow throughout pregnancy.

The objective of the present study was to fabricate and use a bovine liver complementary DNA (cDNA) microarray to profile genome-wide gene expressions in the liver of cow throughout pregnancy. A cDNA library was prepared from liver total RNA collected from cows during estrous cycle and pregnancy, and from fetuses at different stages of pregnancy. The sequenced clones were compiled and annotated by basic local alignment search tool (BLASTn) and spotted onto glass slides. The annotated liver array represented 2675 genes. Of which, 1442 were known genes while 617 sequences had matches with sequences found in expressed sequence tags databases. In addition, 616 unknown sequences were found and these sequences may possibly be identified as candidates for novel bovine genes. For gene expression profiling studies, total RNA from livers of cows slaughtered on days 19, 27-28, 49-58, 150, and 245 of pregnancy (test RNAs) was separately reverse transcribed and labeled with either cyanine 5-fluorescent dye (Cy5) or Cy3. The test samples were individually compared with liver total RNA collected from nonpregnant cycling cows (control RNA) after reverse transcription and labeling with the opposite dye following a two-color hybridization method. After scanning, image acquisition, and normalization, genes that showed either more than 1.5-fold (test/control) induction or repression were selected for further analyses. Hierarchical clustering algorithm showed a clear induction of most liver genes on days 27-28 of pregnancy. Self-organizing maps algorithm identified groups of genes whose differential expression patterns were similar across pregnancy. In conclusion, we described fabrication of a bovine liver cDNA microarray, and demonstrate, for the first time, differential expression patterns of a large number of coregulated liver genes in parallel throughout pregnancy in the bovine.

Algorithms↗