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Biomedical subjects

T Suda

Publications and source records attributed to T Suda.

At least 415 records · Page 23Linked to original sources
PubMed1994

Biological activities of 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 and its 24-epimer.

Biological activities of two epimeric 24-fluorinated vitamin D-2 analogs, 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 [24-F-1,25-(OH)2D2] and its 24-epimer [24-epi-24-F-1,25-(OH)2D2], were studied and compared with 1 alpha,25-dihydroxyvitamin D-3 [1,25-(OH)2D3] and 1 alpha,25-dihydroxyvitamin D-2 [1,25-(OH)2D2]. 24-F-1,25-(OH)2D2 was nearly as active as 1,25-(OH)2D3 and 1,25-(OH)2D2 both in regulating calcium metabolism in vivo including bone mineral mobilization and intestinal calcium transport and in inducing differentiation of HL-60 cells. While 24-epi-24-F-1,25-(OH)2D2 showed distinct properties in these two types of the actions. Though the 24-epimer was nearly as potent as 1,25-(OH)2D3 in inducing differentiation of HL-60 cells, it showed little activity in regulating calcium metabolism in vivo. The fluorine atom introduced at the 24-position of either 1,25-(OH)2D2 or its 24-epimer had no potentiating effect. This is in sharp contrast with the cases of 24- and 26,27-multifluorinated analogs of active vitamin D-3.

Animals↗
PubMed1994

Central administration of alpha-melanocyte-stimulating hormone inhibits corticotropin-releasing factor release in adrenalectomized rats.

To determine if there is a short negative feedback effect of hypothalamic ACTH-related peptides on corticotropin-releasing factor (CRF) release in vivo, we examined the effect of cerebroventricular injection of alpha-melanocyte-stimulating hormone (alpha MSH) on ACTH levels in plasma and the anterior pituitary and CRF levels in the median eminence of the hypothalamus in adrenalectomized or sham-operated rats under pentobarbital anesthesia. alpha MSH did not affect basal ACTH or CRF levels in sham operated rats. However, elevated plasma ACTH levels and CRF levels in the median eminence were decreased by central administration of alpha MSH in adrenalectomized rats. These results suggest that there is a short negative feedback effect of alpha MSH on CRF release and it appears only in the absence of a long negative feedback effect of glucocorticoids.

Adrenalectomy↗
PubMed1994

Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells.

To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca-1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence-activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v-SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.

Amino Acid Sequence↗
PubMed1994

Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene.

The 5'-flanking region of the rat vitamin D3 24-hydroxylase (P450cc24) gene was examined and a vitamin D-responsive element (VDRE) responsible for the 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) enhancement was identified. Unidirectional deletion analyses of the 5'-flanking region indicated that the region [-167/-102] is involved in vitamin D responsiveness. Further functional analyses showed that the segment [-204/-129] conferred the hormone responsiveness in an orientation-independent manner when it was placed upstream to the heterologous thymidine kinase promoter or the rabbit beta-globin promoter. The segment [-204/-129] contained two direct repeat motifs homologous to other VDREs found in the osteocalcin and osteopontin genes. Synthetic oligonucleotides containing the putative VDRE were used for functional analyses and gel mobility shift assays. The proximal [-151/-137], but not the distal [-169/-155] direct repeat activated the transcription in response to 1,25-(OH)2D3 through the beta-globin promoter. Furthermore, the proximal direct repeat formed a complex with the vitamin D receptor and a nuclear accessory factor(s) from COS cells (or retinoid X receptor) in the presence of 1,25-(OH)2D3. These results indicate that a direct repeat motif, AGGTGAgt-gAGGGCG, located at -151 base pairs upstream in the antisense strand binds to a heterologous dimer consisting of the VDR occupied with 1,25-(OH)2D3 and the nuclear accessory factor and that it plays a critical role in mediating the vitamin D enhancement of the rat P450cc24 gene expression.

Animals↗
PubMed1994

Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.

Mice homozygous for lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) develop lymphadenopathy and suffer from autoimmune disease. The lpr mice have a mutation in a cell-surface protein, Fas, that mediates apoptosis. Fas ligand (FasL) is a tumor necrosis factor (TNF)-related type II membrane protein and binds to Fas. Here, mouse Fasl gene was isolated and localized to the gld region of mouse chromosome 1. Activated splenocytes from gld mice express Fasl mRNA. However, FasL in gld mice carries a point mutation in the C-terminal region, which is highly conserved among members of the TNF family. The recombinant gld FasL expressed in COS cells could not induce apoptosis in cells expressing Fas. These results indicate that lpr and gld are mutations in Fas and Fasl, respectively, and suggest important roles of the Fas system in development of T cells as well as cytotoxic T lymphocyte-mediated cytotoxicity.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of the Fas-ligand that induces apoptosis.

Fas is a 45-kD cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family, and transduces the signal for apoptosis. The cytotoxic T lymphocyte (CTL) hybridoma, PC60-d10S requires the presence of Fas on target cells to induce cytolysis in target cells. This CTL cell line was weakly but specifically stained by a chimeric protein that consisted of the extracellular domain of mouse Fas and the Fc portion of human immunoglobulin G1 (mFas-Fc). Moreover, mFas-Fc inhibited the cytotoxic activity of PC60-d10S. Sublines of d10S that were stained intensively by mFas-Fc were isolated by repetitive fluorescence-activated cell sorter sorting. A cell-surface protein of about 40 kD was specifically precipitated by mFas-Fc from the lysates of these sublines. This protein was homogeneously purified by sequential affinity chromatographies using mFas-Fc and concanavalin A beads. The purified protein exhibited cytotoxic activity against cells expressing Fas but not to the cells which do not express Fas. These results indicated that the 40-kD membrane glycoprotein expressed on PC60-d10S cells is the Fas-ligand that induces the apoptotic signal by binding to Fas.

Animals↗
PubMed1994

The role of gravity in chick embryogenesis.

Thirty fertilized chick eggs preincubated for 0, 7 and 10 days on earth (10 eggs each) were flown in the space shuttle 'Endeavour' and further incubated for 7 days under microgravity. Twenty out of thirty eggs (9/10 ten-day-old; 10/10 seven-day-old; 1/10 zero-day-old) were recovered alive after landing. The only living embryo of the zero-day-old group died 24 days after launch, and was comparable to a 16-day-old embryo. The high mortality of the 0-day-old eggs appeared to be related to the specific inner structure of the egg. Simulation experiments performed on earth indicated that when yolk stayed in the albumen for more than 2 days, most of the embryos died. The subtle difference in specific gravity between the yolk (1.029) and albumen (1.040) plays a critical role in early chick embryogenesis.

Animals↗
PubMed1994

Sequence analysis of the proximal promoter region of the human alpha-fetoprotein gene in hepatocellular carcinoma.

We have examined whether or not mutations exist in the proximal promoter region of the human alpha-fetoprotein (AFP) gene in the hepatocellular carcinoma (HCC) tissue. Genomic DNA was extracted from four patients: one HCC tissue, one HCC and its corresponding non-cancerous (cirrhosis) tissues, one liver cirrhosis (LC) tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. Serum concentrations of AFP in the patients ranged from less than 5 to 10,138 ng/ml. Nucleotide sequence was determined by direct sequencing using a single-stranded DNA template that was produced first through the polymerase chain reaction (PCR) amplification and then asymmetric PCR. In one HCC tissue taken from the patient with a high concentration of serum AFP, nucleotides different from published ones were detected at -120 and -113. These changes, however, probably reflect a DNA polymorphism, because peripheral blood leukocytes of the same patient had the same changes. Including this patient, no mutations in the region from -160 to -10 were detected in the HCC specimens we have examined. These results suggest that the extremely proximal promoter region of the AFP gene where glucocorticoid-responsive element and HNF-1 binding sites exist is not responsible for the re-expression of AFP in HCC.

Adult↗
PubMed1994

[Synthesis and release of CRF and ACTH in ectopic CRF/ACTH-producing tumors].

To differentiate between ectopic ACTH syndrome and Cushing's disease, we examined the gene expression of CRF, POMC and glucocorticoid receptor in pituitary adenomas and in ectopic ACTH-producing tumors. CRF increased plasma ACTH levels in all patients with Cushing's disease and in some patients with ectopic ACTH syndrome whose tumors contained CRF and CRF mRNA. In CRF non-responders, no CRF was detected in tumors that contained no CRF mRNA or contained only long-size CRF mRNA. Dexamethasone (Dex) decreased plasma ACTH levels in all patients with Cushing's disease and in the patients with ectopic ACTH-producing bronchial carcinoid. These tumors contained glucocorticoid receptor mRNA. CRF increased and Dex decreased ACTH release and POMC mRNA levels in pituitary adenoma and bronchial carcinoid cells. PMA increased POMC mRNA levels only in carcinoid cells. These results reveal characteristics of ectopic ACTH-producing tumors: long-size CRF mRNA, PMA-induced POMC gene expression, two ectopic ACTH syndrome subtypes (tumors containing ACTH with CRF and tumors without CRF), and Dex-induced decrease in ACTH release and POMC mRNA levels in some bronchial carcinoid.

ACTH Syndrome, Ectopic↗
PubMed1994

Expression of sialosyl-Tn in colony-forming unit-erythroid, erythroblasts, B cells, and a subset of CD4+ cells.

The epitopes Tn and sialosyl-Tn are expressed on erythrocytes of individuals with a very rare blood group, who often suffer from "Tn syndrome." We surveyed expression of Tn and sialosyl-Tn in normal blood cells, malignant transformed cells, and progenitor stem cells from bone marrow (BM). An anti-Tn antibody, IE3, and an anti-sialosyl-Tn antibody, TKH2, were used in this study. TKH2 reacted with erythroblasts, B cells, and a subset of CD4+ cells; but not with erythrocytes. Erythroblastic cell lines (K562, HEL, and UT7/EPO) and B-cell lines (Daudi, Raji, and B-cell lines transformed by Epstein-Barr virus) showed reactivity to TKH2. Similar results from the reactivity of TKH2 with transformed cells from leukemia patients and lymphoma patients were obtained; TKH2 reacted with blasts from erythroleukemia (M6; for 4 of 4 cases) and with lymphocytes from B-cell chronic lymphocytic leukemia (3 of 3), B-cell lymphoma (5 of 5), and CD4+ adult T-cell leukemia (4 of 4), but did not react with blasts from acute myeloid leukemia (M0 to M5; 0 of 22) or acute lymphoid leukemia (B-lymphoid leukemia, 0 of 11; T-lymphoid leukemia, 0 of 2; undifferentiated leukemia, 0 of 1). IE3 did not react with all of the tested cells. CD2-CD19-TKH2+ normal BM cells (BMC) contained blasts and various maturation stages of erythroblasts. The TKH2+ cells produced a large number of colony-forming unit-erythroid (CFU-E) colonies, whereas they produced a small number of burst-forming unit-erythroid colonies and CFU-granulocyte-macrophage colonies. CD34+ normal BMC did not express Tn and sialosyl-Tn. These findings suggest that sialosyl-Tn expresses in CFU-E to erythroblasts.

Antibodies, Monoclonal↗
PubMed1994

Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization.

The expression of the mRNAs for osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP) was studied by in situ hybridization during the healing process of an experimental fracture in adult rat femora. At day 1 postoperatively, ON mRNA was detected in the proliferating periosteum. At day 3, ON, OPN, and OC mRNAs were detected in woven bone. From day 5, MGP and ON mRNAs were detected in the immature chondrocytes. From day 7, ON, OPN, and OC mRNAs were detected in the osteoblastic cells in newly formed endosteal trabecular bone. OPN mRNA was also detected in some of the osteocytes in trabecular bone. From day 14, OPN and MGP mRNAs were detected in newly formed periosteal hypertrophic chondrocytes, and the ON, OPN, and OC mRNAs were detected in osteoblastic cells in newly formed periosteal trabecular bone. Although the cell types that expressed each mRNA in fractured bones were similar to those in embryonic bones, the time course of these mRNA expression in fractured bones was different from that in embryonic bones. We considered that this system is useful to investigate the phenotypic change in osteogenic and chondrogenic lineage cells that appears during fracture healing at the molecular level.

Animals↗
PubMed1994

Glucocorticoid regulation of calcitonin receptor in mouse osteoclast-like multinucleated cells.

Abundant multinucleated cells (MNCs) are formed in cocultures of mouse osteoblastic cells and marrow cells in the presence of 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3], and these cells have the properties of osteoclasts (OCs). In this study using the mammalian OCs, we tried to clarify the role of glucocorticoids (GCs) in calcitonin receptors (CTR) and CT-responsive cAMP production in OCs. Dexamethasone (DEX) dose and time dependently enhanced the specific binding of [125I]salmon calcitonin (sCT). When the MNCs were preincubated with DEX for 24 h, the effect was evident at 10(-9) M and the maximum effect was obtained at 10(-7) M. The effect developed over 12-48 h at doses of 10(-9) and 10(-6) M DEX. The numbers of CTR-positive mononuclear cells and MNCs were not altered by the DEX treatment. Prednisolone and triamcinolone reproduced the DEX effect, but 17 beta-estradiol, progesterone, testosterone, aldosterone, and 1 alpha, 25(OH)2D3 did not. RU486, a GC receptor antagonist, attenuated the effect of DEX to enhance the specific binding of [125I]sCT. From a Scatchard plot analysis, DEX enhanced CTR number (212 +/- 64%) with a minimal change in the affinity to sCT. Autoradiographic studies using [125I]sCT showed that DEX enhanced the density of the grains on the tartrate-resistant acid phosphatase (TRAP)-positive MNCs and mononuclear cells, but not on other types of cells. DEX preincubation also enhanced sCT-stimulated but not prostaglandin E2- or forskolin-stimulated cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Postmitotic osteoclast precursors are mononuclear cells which express macrophage-associated phenotypes.

We previously demonstrated that osteoclast-like multinucleated cells were formed within 6 days in cocultures of mouse osteoblastic cells and spleen cells in response to 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] which was added together with hydroxyurea on Days 4-6 (final 2 days of the 6-day coculture period). Using this coculture system, chronological changes of macrophage-associated phenotypes such as nonspecific esterase (NSE) and antigens to Mac-1, Mac-2, and F4/80 were examined in postmitotic osteoclast precursors during differentiation into osteoclasts induced by 1 alpha,25(OH)2D3 (10 nM) added on Day 4. Osteoclast differentiation was assessed by examining expression of calcitonin receptors (CTRs) by autoradiography using 125I-labeled salmon CT. CTRs were first detected on small mononuclear cells within 12 hr after adding 1 alpha,25(OH)2D3. The number of CTR-positive mononuclear cells attained a maximum at 24 hr and decreased thereafter. CTR-positive multinucleated cells were first observed at 24 hr and reached a maximum population at 48 hr. All CTR-positive cells showed tartrate-resistant acid phosphatase activity (a marker enzyme of osteoclasts). Most of the CTR-positive mononuclear cells which appeared at 12 hr were positive for NSE and antigens to Mac-1 and Mac-2, but negative for F4/80 antigen. The proportion of CTR-positive cells expressing NSE and Mac-1 to total CTR-positive mononuclear cells decreased time dependently. Like authentic osteoclasts, CTR-positive multinucleated cells were negative for NSE and antigens to Mac-1 and F4/80, but positive for Mac-2. These results indicate that postmitotic osteoclast precursors are mononuclear phagocytes with macrophage-associated phenotypes, some of which disappear rapidly during their differentiation into osteoclasts.

Acid Phosphatase↗
PubMed1994

Membrane-associated interleukin-1 promotes osteoclast-like cell formation in vitro.

The effect of paraformaldehyde-fixed murine macrophage P388D1 cells on osteoclast-like cell formation was investigated in mouse marrow cultures. When mouse marrow cells were cocultured for 8 days with paraformaldehyde-fixed P388D1 cells stimulated with lipopolysaccaride (LPS), many tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells were formed. Non-stimulated, and paraformaldehyde-fixed P388D1 cells did not induce the formation of TRACP-positive multinucleated cells. Salmon calcitonin and indomethacin strongly inhibited the paraformaldehyde-fixed, LPS-stimulated P388D1 cells-induced TRACP-positive multinucleated cell formation. Monospecific anti-mouse recombinant IL-1 alpha serum inhibited TRACP-positive multinucleated cell formation in the presence of paraformaldehyde-fixed P388D1 cells stimulated with LPS. These results suggest that membrane-associated IL-1 on macrophages is responsible for the resorptive effect of paraformaldehyde-fixed P388D1 cells stimulated with LPS.

3T3 Cells↗
PubMed1994

Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage.

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.

Alkaline Phosphatase↗
PubMed1994

Novel mouse microsatellites: primer sequences and chromosomal location.

Sixty-nine sequences containing microsatellites were determined by analysis of clones from a pUC118 library of total genomic mouse DNA. These sequences were examined for size variation using polymerase chain reaction and gel electrophoresis. Fifty-one of them showed allelic variations between C57BL/6 and MSM, the two strains used for genetic mapping. Hence, their chromosomal location was determined using a panel consisting of 131 backcross mice that had been typed with 85 anchor loci. The microsatellites were distributed to most chromosomes except for chromosomes 16 and 19. These novel markers with defined locations are useful in linkage and genome mapping studies.

Animals↗
PubMed1994

Human Fas ligand: gene structure, chromosomal location and species specificity.

Fas ligand (FasL) is a 40 kDa type II membrane protein belonging to the tumor necrosis factor family, which induces apoptosis by binding to its receptor, Fas. In this report, we isolated the chromosomal gene for human FasL. The human FasL gene consists of approximately 8.0 kb and is split into four exons. The human FasL gene was mapped on chromosome 1q23 by in situ hybridization against human metaphase chromosomes. Human FasL cDNA was isolated by the reverse polymerase chain reaction of mRNA prepared from human activated peripheral blood lymphocytes. Human FasL is a type II membrane protein consisting of 281 amino acids with a calculated M(r) of 31,759. It has an identity of 76.9% at the amino acid sequence level with mouse FasL. Both human and mouse recombinant FasL expressed in COS induced apoptosis in the cells expressing either human Fas or mouse Fas, indicating that FasL fully cross-reacts between human and mouse. A comparison of human and mouse FasL chromosomal genes indicated that a approximately 300 bp sequence upstream of the ATG initiation codon is highly conserved between them. Several transcription cis-regulatory elements such as SP-1, NF-kappa B and IRF-1 were recognized in this region.

Adult↗