Biomedical subjects

R Sasada

Publications and source records attributed to R Sasada.

At least 19 recordsLinked to original sources

PubMed2001

Novel betacellulin derivatives. Separation of the differentiation activity from the mitogenic activity.

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of beta-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into beta-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.

Amino Acid Sequence↗

PubMed2001

Immunohistochemical localization of Betacellulin, a member of epidermal growth factor family, in atherosclerotic plaques of human aorta.

Betacellulin (BTC), a new member of the EGF family, has been reported to be a potent mitogen for rat vascular smooth muscle cells (SMCs). BTC mRNA is known to be expressed in several human organs. However, the localization of BTC in human vascular tissues has not yet been clarified. We investigated whether or not BTC protein is involved in the pathogenesis of human atherosclerosis. Recombinant human BTC showed a mitogenic activity on cultured human aortic SMCs by measuring [3H]thymidine incorporation. The immunohistochemical localization of BTC, SMCs, macrophages, EGF receptors and ErbB4 was examined in autopsied human aortas. BTC was detected in both intimal and medial SMCs of the aortic wall. The percentage of BTC-positive medial SMCs in early types of atherosclerotic lesions decreased with age, but in adult, it was significantly higher in advanced types than in early types of atherosclerotic lesions. BTC-positive SMCs were predominantly localized in the medial side of the intima. Furthermore, numerous BTC-positive SMCs and macrophages were observed around the core lesion of atherosclerotic plaques. Receptors for BTC, EGF receptor and ErbB4, were expressed on SMCs, suggesting that BTC is associated with EGF receptor family-mediated signaling. BTC is produced in human aortic tissue and might play important roles in atherogenesis.

Adolescent↗

PubMed2000

Molecular cloning and expression of rat betacellulin cDNA.

The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.

3T3 Cells↗

PubMed2000

Recombinant human betacellulin promotes the neogenesis of beta-cells and ameliorates glucose intolerance in mice with diabetes induced by selective alloxan perfusion.

Betacellulin (BTC), a member of the epidermal growth factor family, is expressed predominantly in the human pancreas and induces the differentiation of a pancreatic acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC has a physiologically important role in the endocrine pancreas. In this study, we examined the in vivo effect of recombinant human BTC (rhBTC) on glucose intolerance and pancreatic morphology using a new mouse model with glucose intolerance induced by selective alloxan perfusion. RhBTC (1 microg/g body wt) or saline was injected subcutaneously every day from the day after alloxan treatment. The intraperitoneal glucose tolerance test revealed no difference between rhBTC-treated and rhBTC-untreated glucose-intolerant mice at 2-4 weeks. However, glucose tolerance was significantly improved and body weight was significantly increased in rhBTC-treated mice compared with untreated mice at 8 weeks. Islet-like cell clusters, consisting mainly of beta-cells, were increased in the pancreas and were localized in contact with the ductal lining cells and sometimes with acinar cells. In conclusion, administration of rhBTC improved glucose tolerance in this mouse model by increasing beta-cell volume, primarily through accelerated neogenesis from ductal lining cells.

Animals↗

PubMed1999

Processing and juxtacrine activity of membrane-anchored betacellulin.

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells.

3T3 Cells↗

PubMed1999

Immunohistochemical localization of betacellulin, a new member of the EGF family, in normal human pancreas and islet tumor cells.

Betacellulin (BTC) purified from mouse beta cell tumor (betaTC-3) is a new member of the epidermal growth factor (EGF) family which can bind receptor tyrosine kinase, EGF receptor (erbB1) and erbB4. It has been demonstrated that proBTC mRNA was abundantly expressed in human pancreas tissue, and that BTC converted amylase-secreting rat acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC might be important for the growth and/or differentiation of islet cells. However, the cell type producing BTC in the pancreas has not been clarified. In this study, we examined the localization of BTC in human pancreas and islet cell tumors. Immunohistochemistry using specific antibodies to human BTC revealed that this protein was produced in alpha cells and duct cells, and probably in beta cells in normal adult pancreas. Furthermore, strong immunoreactivity to BTC was detected in primitive duct cells of the fetal pancreas, and both insulinoma and glucagonoma cells also showed positive immunoreactivity to BTC. EGF receptor (erbB1) and erbB4 were expressed mainly in islet and duct cells, and duct cells, respectively. These results demonstrate the localization of BTC and its receptors, and suggest that BTC may be one of the factors that have physiologically important roles such as growth and differentiation of islet cells in the human pancreas.

Adenoma, Islet Cell↗

PubMed1998

BALB/c 3T3 cells co-expressing FGF-2 and soluble FGF receptor acquire tumorigenicity.

The physiological significance of the soluble fibroblast growth factor (FGF) receptors is not clear yet although they are present in blood, vitreous fluid and in the extracellular matrix of vascular endothelial cells. A hypothesis that they might help FGF-2 release from cells is very interesting because FGF-2 does not have clear secretion signal and the mechanism of the secretion of FGF-2 is still unclear. Single overexpression of FGF-2 is related neither to the secretion potential of the molecule nor to the tumorigenicity of the cells. In this report, BALB/c 3T3 cells transformed with the full length of human FGF-2 cDNA are further transformed with the cDNA coding the extracellular domain of human FGF receptor 1. The obtained transformants co-expressing FGF-2 and soluble FGF receptor are highly tumorigenic in nude mice, while the parental cells do not show any tumorigenicity. In the conditioned medium of the double-transformants, FGF-2 is immunologically detected. These results suggest that naturally produced soluble form of FGF receptor supports the release of FGF-2 from the cells and that over-expression of these two molecules leads to induce the malignant tumours in vivo.

3T3 Cells↗

PubMed1997

Protective effect of transfection with secretable superoxide dismutase (SOD) (a signal sequence-SOD fusion protein coding cDNA) expression vector on superoxide anion-induced cytotoxicity in vitro.

For ex vivo gene therapy, superoxide dismutase (SOD) must be secreted into the extracellular space and delivered to damaged cells. Recombinant DNA technique can be used to produce a secretory protein that is fused to a non-secretory protein and a signal peptide of another secretory protein gene. We constructed a secretable SOD eukaryotic expression vector which expresses human SOD cDNA by fusing it to the signal peptide DNA sequence of the human interleukin-2 (IL-2) gene. The ILSOD cDNA constructed by PCR-based gene expression was ligated into the multicloning site of the pRc/CMV plasmid (pRc/CMV-ILSOD). Rat lung epithelial like cells (L2 cells) were transfected with pRc/CMV-ILSOD by lipofection. The extracellular SOD activity of ILSOD-L2 cells (transfected cells with pRc/CMV-ILSOD) was 3 times as high as that of host cells. We used the xanthin (X)/xanthin oxidase (XO) system to produce superoxide anions at the extracellular space. We initially investigated the direct cytotoxicity of superoxide anions upon cells. Host and ILSOD-L2 cells were killed by using X/XO, although the sensitivity of the ILSOD-L2 cells to X/XO induced cytotoxicity was significantly decreased compared with that of host cells. The production of lipid peroxidated substances in the host in the presence of X/XO increased to about twice the control (absence of X/XO) level. However, that of ILSOD-L2 cells did not change in the presence of X/XO. Therefore, ILSOD-L2 cells were resistant to X/XO induced lipid peroxidation. These findings indicated that ILSOD gene transfection protected against direct oxidant stress by X/XO. We then investigated the effect of extracellular SOD secreted from ILSOD-L2 cells on extracellular superoxide anion induced cytotoxicity in normal cells. The conditioned media of host cells had no significant effect upon X/XO induced cytotoxicity. However, the conditioned media of ILSOD-L2 cells protected against X/XO induced cytotoxicity. Furthermore, the conditioned medium of ILSOD-L2 cells was more effective than that of host cells against the production of lipid peroxidated substances by normal cells under conditions of oxidative stress. These results indicated that non-secretable protein could be delivered to target cells by means of DNA engineering. This strategy could thus provide an ex vivo means of applying gene therapy using non-secretable proteins.

Animals↗

PubMed1996

Human betacellulin, a member of the EGF family dominantly expressed in pancreas and small intestine, is fully active in a monomeric form.

Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine. This finding suggests that BTC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family. To clarify this function, the released form of human BTC has been expressed in E.coli, purified, and characterized. The recombinant human BTC was produced as an inclusion body. This material was dissolved in guanidine-HCl under reducing conditions, refolded, and purified through sequential liquid chromatography. Purified BTC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide. However, chemical analysis failed to show a covalently linked dimer. The molecular mass of BTC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer. Recombinant BTC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF On the other hand, BTC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor.

3T3 Cells↗

PubMed1995

A human monoclonal antibody to a human self-antigen, CD2 derived from human peripheral blood lymphocytes engrafted in SCID mice.

To establish human hybridoma lines, production of human immunoglobulin (Ig) and behavior of the implanted human peripheral blood lymphocytes (PBL) were characterized in severe combined immunodeficiency (SCID) mice. Human PBL from healthy donors were injected into the peritoneal cavity of SCID mice, and they were immunized with self-antigen, CD2. CD45+ cells (human PBL) migrated to lymphoid tissues in the mice as early as 4 days, accounting for more than half the lymph node cells and thymocytes. The number of cells releasing human IgG specific to the antigen increased 3.5 weeks after immunization without the usual constraint that production of the IgG, an autoantibody, is prohibited by immunological tolerance in humans. Therefore, we established several human hybridomas secreting human IgG to CD2, since splenocytes and lymph node cells from the implanted SCID mice at 3.5 weeks were fused with a human B lymphoblastoid cell line. A human anti-CD2 monoclonal antibody (MAb) was confirmed to bind to natural CD2 on human T cells by flow cytometric analysis. The epitope for the MAb was identical with a portion that the ligand LFA-3 binded, so that the MAb might reduce the inflammatory reaction caused by preventing activation of human T cells. Here, we report that the human immune system could be reconstituted in SCID mice to develop human hybridomas producing human MAb to a human self-antigen.

Animals↗

PubMed1995

Nitric oxide mediates cytotoxicity and basic fibroblast growth factor release in cultured vascular smooth muscle cells. A possible mechanism of neovascularization in atherosclerotic plaques.

To define the pathophysiological role of nitric oxide (NO) released from vascular smooth muscle cells (VSMC), we examined whether NO released from VSMC induces cytotoxicity in VSMC themselves and adjacent endothelial cells (EC) using a coculture system. Prolonged incubation with interleukin-1 (IL-1) induced large amounts of NO release and cytotoxicity in VSMC. NG-Monomethyl-L-arginine, an inhibitor of NO synthesis, inhibited both NO release and cytotoxicity induced by IL-1. In contrast, DNA synthesis in cocultured EC was not inhibited but rather stimulated by prolonged incubation with IL-1 or sodium nitroprusside (SNP), a NO donor. However, IL-1 and SNP did not stimulate but inhibited DNA synthesis in EC alone. On the other hand, conditioned medium from VSMC incubated for a long period with IL-1 or SNP stimulated DNA synthesis in EC alone. Furthermore, the concentration of basic fibroblast growth factor in the conditioned medium was increased and correlated with the degree of cytotoxicity in VSMC. These results indicate that NO released from VSMC induces VSMC death, which results in release of basic fibroblast growth factor, which then stimulates adjacent EC proliferation. Thus, NO released from VSMC may participate in the mechanism of neovascularization in atherosclerotic plaques.

Animals↗

PubMed1994

Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction.

Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BTC precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1b and BTC-2, were resolved by cation-exchange and size-exclusion column chromatography. Physicochemical analysis has revealed that BTC-1a represents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears to be a truncated molecule lacking 12 amino acid residues from the amino terminus of BTC-1a. BTC-2 was found to be a 50-amino acid molecule (Arg62 to Tyr111) that corresponds to the epidermal growth factor (EGF) structural unit. The biological activities of these BTC molecules were essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cell proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC and EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor molecules with equivalent avidity. The Kd value of EGF receptor (EGFR) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, human mammary carcinoma MDA-MB-453 cells, which express multiple members of the EGFR family, were found to possess 2.7 x 10(3) BTC binding sites/cell, and the binding was readily quenched by EGF. These results suggest that the primary receptor for BTC is EGFR.

3T3 Cells↗

PubMed1994

Purification and characterization of biologically active recombinant human neurotrophin-3 produced by expression of a chimera gene in Chinese hamster ovary cells.

In order to obtain high-level expression of recombinant human neurotrophin-3 (NT-3), we constructed several types of expression plasmids and examined several cell lines for expression of the human NT-3 gene. The highest level production of the recombinant protein was attained in Chinese hamster ovary cells transfected with an expression plasmid that contains a chimera gene encoding the human nerve growth factor (NGF) prepro-region and human NT-3 mature-region under control of a murine leukemia virus-derived long terminal repeat (MuLV-LTR). This cell line can produce more than 1 mg recombinant human NT-3/1 conditioned medium. The recombinant protein was purified to apparent homogeneity with a cation exchange column, a gel filtration column and a reversed-phase HPLC column with a recovery of about 30%. The purified NT-3, at a concentration as low as 0.2 ng/ml, induced neurite out-growth in neurons prepared from 8-day-old chick embryonic dorsal root ganglia; however, it showed little neurotrophic effect on rat PC12 pheochromocytoma cells, which are known to be NGF-responding cells. In addition, this protein promoted colony formation by human peripheral blood lymphocytes in soft agar culture.

Amino Acid Sequence↗

PubMed1994

Antagonism of the human thromboxane A2 receptor by an anti-asthmatic agent AA-2414.

The human thromboxane A2 receptor (TXA2-R)-coding gene was introduced into Chinese hamster ovary cells and a cell line (TCHO-25) stably expressing TXA2-R, at a level of 3 x 10(5)/cell, was obtained. An anti-asthmatic agent AA-2414 [(+-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7- phenylheptanoic acid] competitively inhibited the specific binding of a TXA2 mimic ([3H]U-46619) to the TCHO-25 cells, with an IC50 of 6.0 x 10(-8) M, indicating that the drug is an antagonist of human TXA2-R. The TCHO-25 cells offer a tool for the screening and characterization of human TXA2-R antagonists.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1993

Monoclonal antibodies against human neurotrophin-3.

Hybridomas producing monoclonal antibodies (MoAbs) against human neurotrophin-3 (hNT-3) were established using recombinant hNT-3 produced in CHO cells and E. coli as immunogens. Of the five MoAbs obtained, MoAb 3w3 showed the highest antibody titer and also best neutralized NT-3 activity as measured by the survival of chick embryonic day-8 dorsal root ganglia neurons. A sandwich enzyme immunoassay (EIA) for NT-3 was established with solid phase MoAb 3W3 and the Fab' fragment of MoAb 3W3 conjugated to horseradish peroxidase. The detection limit was 2.7 pg/well of NT-3 and no cross-reactivity with nerve growth factor up to 100 ng/well was observed. Using this EIA system we have screened a variety of cell lines for NT-3 production. Among these tested, only human Burkitt's lymphoma Namalwa cells were found to be producing NT-3.

Animals↗

PubMed1993

Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA.

Previously we showed that motility-related protein (MRP-1) is an antigen recognized by monoclonal antibody (mAb) M31-15 inhibiting cell motility and that the sequence of MRP-1 coincides with that of CD9. In the present study, plasmid was constructed in which human MRP-1/CD9 cDNA is expressed under the control of the Abelson murine leukemia virus promoter sequence. The expression plasmid for MRP-1/CD9 was introduced into Chinese hamster ovary cells, human lung adenocarcinoma cell line MAC10 (MRP-1 positive), and human myeloma cell line ARH77 (MRP-1 negative). All of the MRP-1/CD9 (over)expressing clones obtained from these transfected cells showed suppressed cell motility (penetration and phagokinetic track assays) depending on the degree of expression of MRP-1/CD9. Overexpression of MRP-1/CD9 by MAC10 cells resulted in the suppression of cell motility (maximally 73%) associated with considerable inhibition of the cell growth (maximally 48%). However, the inhibition of the growth of MAC10 cells by mAb M31-15 was < 17% at an antibody concentration of 1-5 micrograms/ml, which inhibits cell motility by > 90%. These results suggest that MRP-1/CD9 directly regulates cell motility and may also affect cell growth. Effects on metastasis by the expression of MRP-1 CD9 were investigated with mouse melanoma BL6 cells-BALB/c nu/nu mouse system. Metastatic potential of all transformants expressing MRP-1/CD9 was lower than that of parent BL6 cells.

Animals↗

PubMed1993

Betacellulin: a mitogen from pancreatic beta cell tumors.

Betacellulin, a member of the epidermal growth factor family, has been identified in the conditioned medium of cell lines derived from mouse pancreatic beta cell tumors. Betacellulin is a 32-kilodalton glycoprotein that appears to be processed from a larger transmembrane precursor by proteolytic cleavage. The carboxyl-terminal domain of betacellulin has 50 percent sequence similarity with that of rat transforming growth factor alpha. Betacellulin is a potent mitogen for retinal pigment epithelial cells and vascular smooth muscle cells.

3T3 Cells↗

PubMed1993

Cloning and expression of cDNA encoding human betacellulin, a new member of the EGF family.

Betacellulin (BTC) is a novel growth factor purified from the conditioned media of mouse pancreatic beta tumor cells and has been found to be a new member of the epidermal growth factor (EGF) family. The cDNA encoding human BTC has been cloned from a cDNA library prepared from human breast adenocarcinoma cell line MCF-7. The nucleotide sequence encodes a polypeptide which consists of 178 amino acid residues including a putative signal sequence, indicating that the structural organization of human BTC is similar to that of mouse BTC. The amino acid sequence of the human BTC precursor protein exhibits 79% similarity with that of the mouse precursor protein. The BTC gene was found to be expressed in several mouse tissues including kidney and liver as well as in a mouse beta tumor cell line and MCF-7 cells, suggesting that BTC might play a physiological role in normal tissues.

Amino Acid Sequence↗