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Biomedical subjects

R Sasada

Publications and source records attributed to R Sasada.

17 recordsLinked to original sources
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↗
PubMed1985

Establishment of hybridomas secreting monoclonal antibodies against C epsilon 2 and C epsilon 4 domains of human IgE.

Three kinds of hybridomas secreting monoclonal antibodies (MAbs) against the epsilon chain of human IgE were constructed by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with human IgE purified from the culture supernatant of U-266 cells. These MAbs were used effectively for the purification and determination of human IgE. The recognition site in the IgE molecule of each antibody was examined by using various epsilon chain fragment peptides produced in Escherichia coli. From these experiments, it was suggested that one recognized C epsilon 2 and the second C epsilon 4. The third did not recognize the C epsilon 1-C epsilon 4 domains of the recombinant epsilon chain from E. coli, although it bound to the epsilon chain of natural human IgE.

Animals↗
PubMed1979

Spontaneous production of a C-type RNA virus in a cell line derived from rat glioma.

The spontaneous production of a rat C-type RNA virus (ACV) in a cultured cell line (AC cells) established from a chemically induced rat glioma was studied. The characteristics of ACV were: morphology typical of C-type RNA virus; buoyant density of 1.15 g/ml in a sucrose density gradient; RNA directed DNA polymerase activity; viral core with a density of 1.28 to 1.30 g/ml; 70S RNA with dimer structure; and structural protein composed of mainly four polypeptides. Kinetical analysis of DNA-DNA hybridization revealed that DNA sequences homologous to DNA transcripts of RNA of ACV were present in rat cells. RNA directed DNA polymerase of ACV partially cross-reacted with antiserum to the polymerase of Rauscher murine leukemia virus. These data suggest that ACV is an endogenous C-type RNA virus of rat origin.

Animals↗
PubMed1978

Biochemical studies on bovine adenovirus type 3. IV. Transformation by viral DNA and DNA fragments.

By the calcium technique, intact DNA of bovine adenovirus type 3 (BAV3) was found to transform A31 cells, a clone of BALB/3T3. Transforming activity was resistant to RNase and Pronase but sensitive to DNase. The efficiency of transformation was approximately 5 to 10 foci per mug of DNA. Attempts were also made to test for transforming activity of BAV3 DNA fragments prepared with restriction endonucleases EcoRI and HindIII. The activity was found to associate exclusively with the EcoRI D fragment mapped in the region of 3.6 and 19.7 units (molecular weight, 3.9 x 10(6)). No transformation could be obtained with three HindIII fragments, J, E, and B, located at the left-hand end of the BAV3 genome. However, the enzymatic joining of J and E fragments (0 to 11.9 map units) with a ligase restored the transforming activity. These results suggest that all the genetic information of BAV3 required for transformation is located in the region between 3.6 and 11.9 units on the viral genome. Some properties of A31 cells transformed by BAV3 DNA EcoRI D fragment (TrD) and the ligated DNA of HindIII J and E fragments (TrJE), as well as those transformed by whole BAV3 DNA (Tr), were examined. As compared to untransformed A31 cells, all the transformed cell lines tested showed rapid growth, high saturation densities, and anchorage-independent growth. Moreover, they contained BAV3-specific T antigen and induced tumors in adult nude and BALB/c mice. These properties of Tr, TrD, and TrJE lines were similar to those of BAV3-transformed cells.

Adenoviridae↗