Biomedical subjects

H Sugino

Publications and source records attributed to H Sugino.

At least 37 records · Page 2Linked to original sources

PubMed2000

Activin A stimulates insulin secretion in cultured human pancreatic islets.

Activin A is a dimeric glycoprotein showing a high sequence homology with transforming growth factor-beta (TGF-beta) and playing autocrine/paracrine actions in reproductive tissues. However, since the synthesis of activin is ubiquitous it may have a role in regulating cell growth and differentiation in several tissues. Previous studies showed that activin A is expressed by insulin-positive B cells of human pancreatic islets, and women with gestational diabetes have higher serum activin A levels than healthy pregnant women at the same gestational age. The present study aimed to evaluate the effect of activin A on insulin secretion from cultured human pancreatic islets. With this purpose human pancreatic islets were incubated with varying concentrations of activin A (0.1 to 10.0 nM). In absence of glucose, activin A did not modify insulin secretion at the different concentrations used. In absence of activin A, 8.3 mM and 16.7 mM glucose significantly increased insulin secretion, with a dose-dependent pattern. In presence of a non stimulatory concentration of glucose (3.3 mM), activin A significantly increased insulin secretion starting from low concentration (0.1 nM). Furthermore, the addition of activin A to 8.3 mM and 16.7 mM glucose induced an additional effect of the dose-dependent glucose-mediated insulin secretion (p<0.001). The present data could support a role for activin A in human endocrine pancreas in modulating insulin response to different glucose concentrations.

Activins↗

PubMed2000

Difference between follistatin isoforms in the inhibition of activin signalling: activin neutralizing activity of follistatin isoforms is dependent on their affinity for activin.

We demonstrate the difference between the follistatin isoforms (FS-288 and FS-315), two activin-binding proteins, in the neutralizing activity for activin signalling. Transcriptional reporter assay using 3TP-Lux, an activin-responsive reporter construct, showed that the inhibitory effect of FS-288 on activin-induced transcriptional response is more potent than that of FS-315. The potency was not influenced by the presence of heparan sulfates, by which FS, in particular FS-288, associates with cell surfaces at a high affinity. Furthermore, FS-288 inhibited the binding of activin to its type II receptor more markedly than did FS-315, as evidenced by surface plasmon resonance and affinity cross-linking experiments. Moreover, the Kd of FS-288 and FS-315 for activin A was estimated to be 46.5+/-0.37 pM and 432+/-26 pM, respectively, by surface plasmon resonance experiments. These results indicate that the different potency between the two FS isoforms in the inhibition of activin activities depends on their affinity for activin A.

Activins↗

PubMed2000

Molecular characterization of a novel nucleolar protein in starfish oocytes which is phosphorylated before and during oocyte maturation.

In response to 1-methyladenine, a maturation-inducing substance, starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown through activation of p34cdc2-cyclin B, which results in the dispersal of the nucleolus. Little information has been elucidated thus far on nucleolar proteins that are phosphorylated by p34cdc2-cyclin B during meiotic maturation. Here, we describe a novel nucleolar protein of the starfish Asterina pectinifera oocyte, which is designated ANO39 and which is phosphorylated during meiotic maturation. A full-length ANO39 cDNA of 2106 base pairs encodes a polypeptide of 346 amino acids having a calculated Mr of 39 005. The amount of ANO39 is kept nearly constant during oocyte maturation and embryogenesis up to the midgastrula stage. The transcript encoding ANO39 was present in growing oocytes but not in full-grown ones, as evidenced by Northern blot hybridization. Ser145 is specifically phosphorylated when ANO39 is incubated in vitro with purified starfish p34cdc2-cyclin B. This phosphorylation site corresponds to that is phosphorylated during meiotic maturation in vivo. Immunoblot analysis using phosphoserine145-specific antibody as a probe revealed that some populations of ANO39 of the immature oocytes at the G2 stage have been already phosphorylated on Ser145 and Ser145 is maximally phosphorylated during meiotic maturation.

Adenine↗

PubMed2000

Effect of activin on production and secretion of prolactin and growth hormone in cultured rat GH3 cells.

OBJECTIVE: To evaluate the effect of the growth factor activin A on the secretion of prolactin (PRL) and GH in cultured GH3 cells. METHODS: The concentrations of PRL and GH secreted from GH3 cells cultured in media with and without activin A were measured by RIA, and the expression of PRL mRNA and GH mRNA were analyzed using the Northern blot method. RESULTS: Activin A significantly inhibited PRL release from GH3 cells cultured for 48h in a dose-dependent manner (activin: 0.3-3nM). The inhibitory effects of 3nM activin A were observed in the culture from 12h to 48h (53.2% of control). Activin A (3nM) also significantly inhibited the expression of PRL mRNA at 24h (33.8% of control). In contrast, activin A significantly stimulated GH release from GH3 cells cultured for 48h in a dose-dependent manner (activin: 0.3-3nM). The stimulatory effect of 3nM activin A was observed in the culture for 48h (157.6% of control). Activin A (3nM) also significantly stimulated the expression of GH mRNA at 24h (183.6% of control). In spite of these significant changes in PRL and GH secretion, pit-1 mRNA levels were not significantly changed by activin A. CONCLUSIONS: These findings indicated that activin A modulates PRL and GH secretion through the regulation of PRL and GH gene transcription in GH3 cells, but that these effects are unrelated to pit-1 gene expression.

Activins↗

PubMed2000

Expression patterns of follistatin and two follistatin-related proteins during mouse development.

We compared the expression patterns of follistatin and two follistatin-related proteins (FRP and m7365) during early mouse development. m7365 is expressed continuously during preimplantation development, in contrast to FRP and follistatin. At early postimplantation stages, follistatin and 7365 are expressed from E6.0, while FRP is detected from E7.5 onwards. Although there is some overlap between the expression of these genes in the primitive streak and somites, their overall expression patterns are distinct.

Animals↗

PubMed1999

Differential inhibition of Smad6 and Smad7 on bone morphogenetic protein- and activin-mediated growth arrest and apoptosis in B cells.

Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-beta family. Here we demonstrate that both Smad6 and Smad7 are human bone morphogenetic protein-2 (hBMP-2)-inducible antagonists of hBMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells. Moreover, we confirmed that the ectopic expressions of Smad6 and Smad7 inhibited the hBMP-2-induced Smad1/Smad5 phosphorylation. We previously reported that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis in HS-72 cells. Interestingly, although mRNA expression of Smad6 was induced by activin A in HS-72 cells, Smad6 showed no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, we found that the ectopic expression of Smad7, but not Smad6, inhibited the activin A-induced Smad2 phosphorylation in HS-72 cells. Thus, Smad6 and Smad7 exhibit differential inhibitory effects in bone morphogenetic protein-2- and activin A-mediated signaling in B lineage cells.

Activins↗

PubMed1999

cDNA cloning and distribution of the Xenopus follistatin-related protein.

Recently, several proteins which have a follistatin module have been isolated. One of them, the follistatin-related protein (FRP), is encoded by TSC-36 (TGF-beta-stimulated clone 36) in mouse, originally isolated as a cDNA clone up-regulated by TGF-beta1 in mouse osteogenic MC3T3E1 cells. To determine the physiological role of FRP in early Xenopus embryonic development, we cloned the Xenopus FRP (xFRP) cDNA. The resulting cDNA clone was a secreted glycoprotein consisting of 299 amino acid residues with about 70% similarity to the mammalian and avian FRPs. Northern blotting analysis revealed that xFRP gene expression started at stage 10, the onset of gastrulation, gradually increased during the blastula and neurula stages and was sustained through the tail-bud stage. Whole-mount in situ hybridization analysis showed the localization of xFRP mRNAs in the Spemann organizer, notochord, neural floor plate, hypochord and somite. The similarities with the pattern of expression of Xenopus follistatin mRNA suggests that xFRP may play a role in neuralization.

Amino Acid Sequence↗

PubMed1999

Effects of activin on hormone secretion by single female rat pituitary cells: analysis by cell immunoblot assay.

We investigated the effect of activin A on secretion of LH, FSH, and prolactin (PRL) by female cultured rat pituitary cells at the single-cell level by means of the cell immunoblot assay. Anterior pituitary cells from 8-week-old female rats were preincubated with or without activin A for 24 h, after which they were monodispersed and immediately used for cell immunoblot assay. The percentages of LH-, FSH- and PRL-immunoreactive cell blots detected were 5.5, 5.3 and 43.1%, respectively, of all pituitary cells applied to the transfer membrane. The percentage of LH-secreting cells and mean LH secretion per cell did not change after treatment with activin. In contrast, activin significantly increased the percentage of FSH-secreting cells and mean FSH secretion per cell to 136.0 and 114. 5% respectively. In addition, activin significantly decreased the percentage of PRL-secreting cells and mean PRL secretion per cell to 52.2 and 72.0% respectively. These results suggest that (1) activin A has effects on female rat pituitary cells that increase not only the amount of FSH secretion per cell but also the number of FSH-secreting cells, and (2) activin A decreases both the amount of PRL secretion per cell and the number of PRL-secreting cells.

Activins↗

PubMed1998

Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo.

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.

Animals↗

PubMed1998

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.

Activin Receptors↗

PubMed1998

Identification of a novel type II activin receptor, type IIA-N, induced during the neural differentiation of murine P19 embryonal carcinoma cells.

We have identified a novel type II activin receptor, called type IIA-N, the expression of which was induced during the neural differentiation of murine P19 embryonal carcinoma cells (P19 cells). P19 cells differentiate into several cell types dependent on the culture conditions. The induction of type IIA-N mRNA occurred predominantly in conjunction with neural differentiation. Sequence analysis of a cDNA clone for type IIA-N indicated that type IIA-N had a 24 bp insertion in the juxtamembrane region of the type IIA activin receptor suggesting that it is an alternative splicing product of the type IIA gene. Type IIA-N was also identified in human and Xenopus, and the amino acid sequences of three species were completely conserved. The expression of type IIA-N mRNA was specifically detected in neuroblastoma cells among several activin responsive cell lines. In vivo expression of type IIA-N mRNA was detected only in the neural tissues such as brain and spinal cord in adult mouse, by RT-PCR. Furthermore, its expression in developing Xenopus embryos was restricted to the neurula and later stages. These results suggest that the expression of type IIA-N is specific to neural cells and mediates neural differentiation-specific activin signaling.

Activin Receptors↗

PubMed1998

Molecular characterization of Xenopus embryo heparan sulfate. Differential structural requirements for the specific binding to basic fibroblast growth factor and follistatin.

Enzymatic elimination of heparan sulfate (HS) causes abnormal mesodermal and neural formation in Xenopus embryos, and HS plays an indispensable role in establishing the embryogenesis and tissue morphogenesis during early Xenopus development (Furuya, S., Sera, M., Tohno-oka, R., Sugahara, K., Shiokawa, K., and Hirabayashi, Y. (1995) Dev. Growth Differ. 37, 337-346). In this study, HS was purified from Xenopus embryos to investigate its disaccharide composition and binding ability to basic fibroblast growth factor (bFGF) and follistatin (FS), the latter being provided in two isoforms with core sequences of 315 and 288 amino acids (designated FS-315 and FS-288) originating from alternative mRNA splicing. Disaccharide composition analysis of the purified Xenopus HS showed the preponderance of a disulfated disaccharide unit with uronic acid 2-O-sulfate and glucosamine 2-N-sulfate, which has been implicated in the interactions with bFGF. Specific binding of the HS to bFGF and FS-288, the COOH-terminal truncated form, was observed in the filter binding assay, whereas HS did not bind to FS-315, indicating that the acidic Glu-rich domain of FS-315 precluded the binding. The binding of the HS to bFGF or FS-288 was markedly inhibited by heparin (HP) and various HS preparations, but not by chondroitin sulfate, supporting the binding specificity of HS. The binding specificity was further investigated using FS-288 and bovine intestinal [3H]HS. Competitive inhibition assays of the HS binding to FS-288 using size-defined HP oligosaccharides revealed that the minimum size required for significant inhibition was a dodecasaccharide, which is larger than the pentasaccharide required for bFGF binding. The binding affinity of FS to HS increased in the presence of activin, a growth/differentiation factor, which could be inactivated by direct binding to FS. These results, taken together, indicate that the structural requirement for binding of HS to bFGF and FS is different. HS may undergo dynamic changes in its structure during early Xenopus embryogenesis in response to the temporal and spatial expression of various growth/differentiation factors.

Activins↗

PubMed1998

Correlation between Bcl-X expression and B-cell hybridoma apoptosis induced by activin A.

In this study, we examined the role of bcl-XL and bcl-XS in apoptotic cell death of HS-72 cells induced by activin A. Immunoblot analysis revealed that a band of Bcl-XL was detected in HS-72 cells cultured with or without activin A. Although untreated HS-72 cells did not express Bcl-XS, the expression of Bcl-XS was significantly increased when cultured with activin A. We also investigated the expression of Bcl-XS and Bcl-XL in HS-72 cells cultured with activin A in the presence of protein kinase C inhibitor 1-(5-isoquinotinesulfonyl)-2-methylpiperazine dihydrochloride (H7), which suppressed apoptosis in HS-72 cells induced by activin A. Exposure to H7 apparently increased the level of Bcl-XL in HS-72 cells cultured with or without activin A. In contrast, no detectable band of Bcl-XS was found in HS-72 cells cultured with activin A and H7. These findings indicate that Bcl-XL upregulation and Bcl-XS downregulation induced by H7 might correlate with the suppression of activin A-induced apoptosis in B-lineage cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

The role of activin type I receptors in activin A-induced growth arrest and apoptosis in mouse B-cell hybridoma cells.

Activins transduce their signals by binding to activin type I receptors and activin type II receptors, both of which contain a serine/threonine kinase domain. In this study, we established stable transfectants expressing two types of activin receptors, ActRI and ActRIB, to clarify the role of these receptors in activin signalling for growth inhibition in HS-72 mouse B-cell hybridoma cells. Over-expression of ActRI suppressed activin A-induced cell-cycle arrest in the G1 phase caused by inhibition of retinoblastoma protein phosphorylation through induction of p21CIP1/WAF1, a cyclin-dependent kinase inhibitor, and subsequent apoptosis. In contrast, HS-72 clones that over-expressed ActRIB significantly facilitated activin A-induced apoptosis. These results indicate that ActRI and ActRIB are distinct from each other and that the ActRI/ActRIB expression ratio could regulate cell-cycle arrest in the G1 phase and subsequent apoptosis in HS-72 cells induced by activin A.

Activin Receptors, Type I↗

PubMed1998

Activin A regulates the production of mature interleukin-1beta and interleukin-1 receptor antagonist in human monocytic cells.

Activin A, a member of the transforming growth factor-beta (TGF-beta) family, is produced by a variety of cells and implicated in the regulation of the reproductive endocrine system, mesoderm induction, and erythropoiesis. In the present study, we showed that activin A inhibited the production of interleukin-1beta (IL-1beta), a potent proinflammatory cytokine, and enhanced the production of IL-1 receptor antagonist (IL-1ra), in activated THP-1 and U-937 human monocytic cells, resulting in the reduction of IL-1 biologic activity. Northern blot analysis revealed that activin A had no effect on mRNA accumulation of IL-1beta and IL-1ra, indicating that activin A regulates IL-1beta and IL-1ra production at a posttranscriptional level. As it is well known that an inactive precursor form of IL-1beta (pro-IL-1beta) is converted to an active mature form (mature IL-1beta), we examined the expression levels of pro-IL-1beta and mature IL-1beta by immunoblot analysis. Although activin A inhibited the production of mature IL-1beta in activated U-937 cells, the relative protein expression of pro-IL-1beta was unaltered by activin A, suggesting that activin A inhibits IL-1beta production by blocking proteolytic conversion of pro-IL-1beta into mature IL-1beta. Taken together, these findings suggest that activin A may function as an anti-inflammatory cytokine by modulating mature IL-1beta and IL-1ra production in inflammatory sites.

Activins↗

PubMed1997

Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent.

A novel sialylphospholipid (SPL) was synthesized from N-acetylneuraminic acid (NeuAc) and phosphatidylcholine (PC) by a chemical and enzymatic method and evaluated as an inhibitor of rotavirus. PC and 1,8-octanediol were conjugated by transesterification reaction of Streptomyces phospholipase D (PLD) under a water-chloroform biphasic system to afford phosphatidyloctanol, which was condensed with a protected 2-chloro-2-deoxyneuraminic acid derivative by using silver trifluoromethanesulfonate as an activator in chloroform and converted, after deprotection, to SPL. Rhesus monkey kidney cells (MA-104) were incubated with simian (SA-11 strain) and human (MO strain) rotaviruses in the presence of SPL, and the cells infected were detected indirectly with anti-rotavirus antibody. SPL showed dose dependent inhibition against both virus strains. The concentrations required for 50% inhibition (IC50) against SA-11 and MO were 4.35 and 16.1 microM, respectively, corresponding to 10(3)- and 10(4)-fold increases in inhibition as compared to monomeric NeuAc.

Animals↗

PubMed1997

Distribution of human endogenous retroviral RTVL-H2 LTR sequences among human chromosomes.

The human genome carries multiple copies of sequences related to endogenous retroviral DNA. We report here the distribution of a new multicopy long terminal repeat (LTR) sequence that has been a part of an endogenous retrovirus-like sequence RTVL-H2. Twenty-four human chromosomes were either separated by flow sorting or by using rodent cells carrying a single human chromosome, and the DNA was subjected to Southern analyses using the RTVL-H2 DNA as a probe. The RTVL-H2 LTRs were distributed among all the human chromosomes, but the density and the profile differed from chromosome to chromosome. The same chromosome obtained from different individuals showed essentially the same chromosome-specific patterns. The distribution of the RTVL-H2 LTRs among different chromosomes did not correlate with the distribution of LTRs from another endogenous retroviral DNA, HERV-A, strongly suggesting that there is no preferred chromosome or a region thereof, for the integration. The possibility of rearrangement or amplification after integration is discussed.

Blotting, Southern↗

PubMed1997

Genomic organization and chromosomal localization of the mouse telencephalin gene, a neuronal member of the ICAM family.

Telencephalin is a cell adhesion molecule belonging to the immunoglobulin (Ig) superfamily, whose expression is restricted to subsets of neurons in the telencephalon, the most rostral segment of brain. Of all the Ig superfamily molecules so far identified, the structure of telencephalin is most closely related to those of intercellular adhesion molecules (ICAMs)-1 and -3. Here we report the cloning, characterization, and chromosomal localization of the mouse telencephalin gene (Tlcn). The Tlcn gene spanned about 6.3 kb and consisted of 11 exons. A signal peptide and individual nine Ig-like domains of telencephalin were encoded by a single exon, while the transmembrane and cytoplasmic regions were fused in a same exon. The primer extension technique was used to establish that the transcription initiation sites were located 92-95 bp upstream from the ATG start codon. DNA sequencing of the 5'-flanking region revealed the presence of a strong initiator element for TATA-less genes, two CAAT boxes, and numerous potential transcription factor binding sites including four E-box and two N-box sequences. Interspecific backcross analysis demonstrated that the Tlcn gene was mapped in the proximal region of mouse chromosome 9 in close vicinity to the Icam-1 gene, suggesting that Tlcn and Icam-1 are derived from a common ancestral gene by gene duplication.

Animals↗