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Biomedical subjects

R Sasada

Publications and source records attributed to R Sasada.

At least 37 records · Page 2Linked to original sources
PubMed1993

Novel secretory heparin-binding factors from human glioma cells (glia-activating factors) involved in glial cell growth. Purification and biological properties.

Growth factors for rat primary glial cells were identified in conditioned medium of a human glioma-derived cell line. The factors, designated glia-activating factors (GAFs), were purified to homogeneity by a combination of heparin affinity chromatography, gel filtration, and high performance liquid chromatography on a heparin affinity column and a C4 reversed-phase column. GAFs could be resolved into three peaks by C4 column chromatography. The M(r) values of these three proteins were estimated to be 30,000, 29,000, and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These M(r) values were in good agreement with the value of 26,000 +/- 3,000 estimated from the elution volume upon gel filtration chromatography under nondenaturing conditions. These data suggested that each of the GAFs consists of a single polypeptide chain and has no subunit structures. These three purified GAFs had almost the same growth-stimulating effect on glial cells in vitro, and the half-maximal dose was around 10(-11) M. Concanavalin A staining and glycopeptide N-glycosidase treatment of GAFs indicated that an asparagine-linked oligosaccharide chain(s) was attached to these three kinds of GAFs. Microsequencing of each GAF revealed a single amino-terminal sequence with no significant homology to any known protein, and the amino-terminal sequence of the 30-kDa GAF included that of the 29-kDa GAF. GAFs also stimulated the cell growth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3 fibroblasts, and PC-12 cells but not that of human umbilical vein endothelial cells.

Amino Acid Sequence↗
PubMed1993

Identification of the functional regulatory region of the neurotrophin-3 gene promoter.

The 5'-flanking region of the human neurotrophin-3 (NT-3) gene was isolated from a human placental genomic library using the oligonucleotide corresponding to the 5'-noncoding region of the NT-3 cDNA as a probe. A 3.8 kbp genomic fragment containing the 5'-flanking region, the first exon and a portion of the first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 nuclease mapping, was located 27 bp downstream from the TATA-like sequence. Several plasmids, in which the NT-3 promoter regions were fused to the chloramphenicol acetyltransferase (CAT) gene, were constructed. Transient expression in human glioma Hs683 cells demonstrated that a fragment of about 0.1 kbp from the transcriptional initiation site was sufficient for promoter activity. While, in human plasma cell leukemia ARH77 cells, in which NT-3 mRNA was not detected, the region upstream from -65 functioned to silence CAT activity. It is suggested that this region contains the transcriptional regulatory element for the specific expression of the NT-3 gene.

Base Sequence↗
PubMed1993

Monoclonal antibodies against hst-1 gene product.

Four monoclonal antibodies (MAbs) against the hst-1 gene product (hst-1 protein) were obtained by a somatic cell hybridization technique. The recognition sites of these MAbs designated HS-131, HS-210, HS-233 and HS-276 on the hst-1 protein were evaluated by competitive binding assay with synthetic polypeptides. HS-131 MAb and HS-276 MAb recognize the epitope located within the 59-73 and the 197-206 amino acid sequences, respectively. The epitopes recognized by HS-210 and HS-233 MAbs could not be determined, but these MAbs showed neutralizing activity against hst-1 protein. Using HS-131 and HS-233 MAbs, a sensitive sandwich enzyme immunoassay (sandwich EIA) has been developed. The assay sensitivity was 1.2-2.5 pg/well of hst-1 protein. Acidic and basic fibroblast growth factors were not cross-reactive up to a concentration of 1 microgram/ml.

3T3 Cells↗
PubMed1993

[Betacellulin: a new growth factor for vascular smooth muscle cells].

Betacellulin, a new member of the epidermal growth factor family, has been purified from the conditioned media of mouse pancreatic beta tumor cells. Purified betacellulin is a single chain glycoprotein with a molecular weight of approximately 32K. This factor is a potent mitogen for retinal pigment epithelial cells and vascular smooth muscle cells. Molecular cloning of the mouse and human cDNAs reveals that the mature form of this factor, composed of 80 amino acid residues, appears to be processed from a larger transmembrane precursor by proteolytic cleavage. Seventy nine % amino acid similarity is present between mouse and human precursor proteins. The carboxyl-terminal domains of both mouse and human betacellulins have a 50% sequence similarity with that of rat transforming growth factor alpha. The betacellulin gene is found to be expressed in several mouse tissues, including kidney, liver and pancreatic beta cells.

Amino Acid Sequence↗
PubMed1991

Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent angiogenic mitogen. To elucidate the effect of bFGF inhibitors in vivo, anti-bFGF immunoneutralizing monoclonal antibody was prepared. One monoclonal antibody against human bFGF, obtained by cell fusion and designated 3H3, completely inhibited bFGF-induced proliferation of human umbilical vein endothelial cells at a concentration of 100 ng/ml. 3H3 did not bind to acidic fibroblast growth factor or HST1 protein, indicating high specificity for bFGF. Furthermore, the immunoneutralizing activity of 3H3 was examined in vivo. K1000 cells (a BALB/c 3T3 transformant in which the leader sequence-fused bFGF gene was transfected) were transplanted s.c. into BALB/c nude mice. Growth of the tumor cells was inhibited by i.v. treatment with 3H3 at a concentration of 200 micrograms/mouse. Histological observation showed that the antitumor effect of 3H3 was due to the inhibition of bFGF-induced angiogenesis. This experiment provides direct causal evidence for the hypothesis that tumor growth is angiogenesis dependent. This finding could also have implications for the development of novel therapeutic approaches to angiogenic solid tumors.

Animals↗
PubMed1991

Two cDNAs encoding novel human FGF receptor.

Two types of cDNAs encoding novel human FGF receptors were isolated. These two cDNAs were found to be closely related to the oncogene bek. Products from these genes were membrane-bound when their cDNAs were transiently expressed in COS cells, whereas products from the regions coding extracellular domains were free of membrane attachment and found in the culture medium.

Amino Acid Sequence↗
PubMed1990

Production, purification and characterization of biologically active recombinant human nerve growth factor.

The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.

Amino Acid Sequence↗
PubMed1990

Carboxyl-terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin.

The carboxyl-terminal sequence of basic fibroblast growth factor (bFGF) is rich in basic amino acid residues, a common characteristic amongst fibroblast growth factors, and is considered to contribute greatly to the binding to negatively charged extracellular matrixes such as heparin. To study the relationship between the affinity for heparin and the carboxyl-terminal structure of bFGF, amino- or carboxyl-terminal truncated molecules were produced in Escherichia coli using recombinant DNA techniques. These terminally truncated bFGFs were applied to a heparin-affinity HPLC column. Truncation of more than six amino acid residues from the carboxyl-terminal made the bFGF produced in E. coli markedly difficult to solubilize and weakened its affinity for heparin, though bFGF having up to 46 amino acids removed showed significant stimulation of the DNA synthesis of BALB/c3T3 cells. This stimulation of the DNA synthesis was also recognized by the bFGF having 40 amino acids removed from its amino-terminal, while the affinity of this peptide for heparin has been shown to be equal to that of the mature bFGF (146 amino acids). These results show that the affinity of bFGF for heparin depends significantly on its carboxyl-terminal structure and that the essential part for receptor binding is present between Asp41 and Ser100. Moreover, it suggests that the Phe139Leu140Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule.

Amino Acid Sequence↗
PubMed1990

Improvement in the antibody productivity of human-human hybridomas by transfection with Tac gene.

The presence of a highly purified recombinant interleukin-2 (rIL-2) increased the production of immunoglobulin (IgM or IgG) by human-human hybridomas to 1.5-2.0 times the production by untreated cells. However, these cells did not react with anti-Tac (IL-2 receptor alpha) antibody. To enhance the response of the hybridoma cells to rIL-2, Tac gene was introduced by co-transfection with Tac gene expression plasmid pTB459 and G418 resistant gene expression plasmid pRSVneo. Tac cDNA transfected hybridoma (HBW-4.16.459-6-126) was induced to produce 6 times as much IgG by rIL-2 as was the control. This antibody production promoting phenomenon mediated by rIL-2 was depressed by anti-Tac antibody.

Antibodies, Monoclonal↗
PubMed1990

Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.

Cell Division↗
PubMed1989

Monoclonal antibodies against human basic fibroblast growth factor.

Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.

Animals↗
PubMed1988

Stabilizing basic fibroblast growth factor using protein engineering.

Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine. The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity. The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations. Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable in maintaining their activity. These facts suggest that this protein has been successfully modified by protein engineering.

Base Sequence↗
PubMed1988

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.

Animals↗
PubMed1988

Secretion of human EGF and IgE in mammalian cells by recombinant DNA techniques; use of a IL-2 leader sequence.

Expression plasmids were constructed containing chemically synthesized human epidermal growth factor (EGF) gene fused in a frame to a leader sequence of human interleukin-2 (IL-2) gene under the control of a viral promoter. COS7 cells transfected with the plasmids synthesized and secreted EGF. Transfection of mouse A9 cells or BALB/3T3 clone A31 cells with the plasmids permitted the isolation of cell lines secreting the product which showed EGF activity. In particular, A31 transformed cells secreting human EGF grew well even in a medium containing a minimal level of serum. Using similar vectors having IgE cDNA (C2-C4) in place of EGF gene, a human IgE Fc fragment was also produced and secreted in mouse cells. These results show that heterologous leader sequences are useful for the expression and secretion of proteins whose genes lack leader sequences.

Animals↗
PubMed1987

Expression of cDNA encoding human basic fibroblast growth factor in E. coli.

The cDNA encoding human basic fibroblast growth factor was expressed in E. coli under the control of trp promoter. Bacterially synthesized hbFGF was highly purified using a heparin affinity HPLC column. By this chromatography, hbFGF was eluted as four distinct forms, which were indistinguishable by SDS polyacrylamide gel electrophoresis, amino acid composition, and partial terminal sequence analysis. These molecules stimulated the growth of fibroblasts and endothelial cells although their specific activities varied. The angiogenesis activity of these molecules was also confirmed.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of cDNA encoding human basic fibroblast growth factor.

A cDNA encoding human basic fibroblast growth factor (bFGF) was isolated from a human foreskin fibroblast cDNA library. The cDNA, 4 kilobases in size, had a coding sequence, 5' and 3' untranslated regions, and a poly(A) chain. Isolation of additional cDNA clones that had a short 3' untranslated region suggested the presence of multiple mRNA forms. By Northern blot analysis, at least five bFGF mRNA species were detected in cultured fibroblast cells. Transfection of the cDNA to COS cells resulted in the detection of mitogenic activity in the culture medium of the transfected cells, suggesting that a part of the synthesized protein might be secreted from cells despite its unusual short signal sequence.

Amino Acid Sequence↗
PubMed1987

The establishment of IL-2 producing cells by genetic engineering.

Expression plasmids containing human interleukin-2(IL-2) cDNA under the control of viral promoters (SV40 early region, MuLV LTR, HTLV-I LTR, and ASV (Y73) LTR) were introduced into TK- mouse L cells and human FL cells to establish IL-2 producing cells. The highest levels of IL-2 producing clones were obtained in TK+ mouse L cells transformed with a recombinant plasmid having MuLV LTR as a promoter, whereas transformed cells of human FL cells (G418r) were revealed to produce IL-2 at the highest level when the cells were transfected with a plasmid containing HTLV LTR as a promoter. These results suggest that these promoter/enhancer regions possess different cell specificities in gene expression. To obtain higher levels of IL-2 production using gene amplification, the hybrid plasmids containing the hamster DHFR and human IL-2 genes were constructed and transfected into DHFR- CHO cells. DHFR+ colonies produced IL-2 at about the same level as that produced by TK+ L cells transformed with the recombinants containing MuLV LTR. Selection of methotrexate-resistant cells resulted in a 5- to 30-fold increase of IL-2 production. These cells produced IL-2 stably for at least 3 months, even in the absence of methotrexate.

Animals↗