Biomedical subjects

T Kajio

Publications and source records attributed to T Kajio.

5 recordsLinked to original sources

PubMed1992

Stabilization of basic fibroblast growth factor with dextran sulfate.

Dextran sulfate protected bFGF from heat and acid inactivation and from proteolytic degradation. The protective effect was stronger than that of heparin which is known as a stabilizer of bFGF. Dextran sulfate and bFGF formed a high molecular weight complex via ionic interaction when mixed together in aqueous solution. The complex was dissociated when the ionic strength was increased and the protective effect was completely abolished. Successive digestion of bFGF with Staphylococcus aureus V8 protease and pepsin followed by affinity chromatography on an immobilized dextran sulfate column and reversed-phase high performance liquid chromatography yielded three positively charged fragment peptides, Tyr24-Phe30, Tyr106-Trp114 and Tyr124-Leu138. These results suggest that dextran sulfate stabilizes bFGF by binding close to the putative heparin binding sites of the bFGF molecule.

Amino Acid Sequence↗

PubMed1992

Quantitative colorimetric assay for basic fibroblast growth factor using bovine endothelial cells and heparin.

An accurate and reproducible colorimetric assay was established to determine the concentration of basic fibroblast growth factor (bFGF) or bFGF-like activity in culture media and biological fluids. Fetal bovine heart endothelial cell line ATCC CRL 1395 was used as the bFGF-dependent cell line. The proliferation-stimulating activity of bFGF was determined by measuring the amount of formazan formed by the mitochondrial enzymes from a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), instead of counting the viable cell numbers or measuring the incorporation of [3H]-thymidine. The addition of 250 ng/mL of heparin to the culture medium resulted in about a tenfold increase in the proliferation-stimulating activity of bFGF and allowed the detection of as low as 10 pg/mL of bFGF. Heparin also resulted in much smaller inter- and intraassay variations. The bFGF concentrations determined by this colorimetric assay correlated well with those determined by both the [3H]-thymidine incorporation assay using BALB/c 3T3 fibroblast cells (r = 0.998) and the cell number count assay (r = 0.996). This assay can be adapted to quantify bFGF or bFGF-like activity in tissue culture media and biological fluids such as plasma and organ extracts.

Animals↗

PubMed1989

Escherichia coli-derived human interferon-gamma with Cys-Tyr-Cys at the N-terminus is partially N alpha-acylated.

Purified preparations of recombinant human interferon-gamma (rIFN-gamma) with Cys-Tyr-Cys at the N-terminus ([ Cys-Tyr-Cys]IFN-gamma) derived from Escherichia coli gave two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two peaks on reversed-phase high-performance liquid chromatography (rpHPLC). In contrast, rIFN-gamma without Cys-Tyr-Cys and rIFN-gamma in which both Cys-1 and Cys-3 were substituted with serine behaved as a single species on both SDS-PAGE and rpHPLC. These results suggest that the N-terminal portion of rIFN-gamma is heterogeneous. To elucidate the structure of the N-terminal portion, the N-terminal peptide preparation was obtained by binding rIFN-gamma to thiopropyl-Sepharose 6B gel with disulfide linkage followed by trypsin digestion and elution with 2-mercaptoethanol. The preparation gave four peaks (NT-1, NT-2, NT-3, and NT-4, in order of elution) on rpHPLC; all four were found to be Cys-1-Lys-9 by amino acid analysis after acid hydrolysis. Various analyses indicate that NT-1 is the intact nonapeptide, that NT-3 and NT-4 are N alpha-formyl and N alpha-acetyl forms of NT-1, respectively, and that NT-2 may be S-blocked at Cys-1. It is concluded that E. coli-derived [Cys-Tyr-Cys]IFN-gamma is partially N alpha-acylated. The data also suggest that N alpha-acylation does not affect the biological activity of [Cys-Tyr-Cys]IFN-gamma.

Amino Acid Sequence↗

PubMed1988

Antibodies against basic fibroblast growth factor inhibit the autocrine growth of pulmonary artery endothelial cells.

Anti-recombinant human basic fibroblast growth factor (rbFGF) antibodies efficiently inhibited the basal proliferation of bovine pulmonary artery endothelial (BAE) cells. The cell-free extract of BAE cells stimulated the proliferation of bovine capillary endothelial cells and this activity was completely abolished by the antibodies. Furthermore, on heparin HPLC the activity was eluted at exactly the same retention time as that for authentic pituitary bFGF. These observations directly indicate that the BAE cells produce bFGF that stimulates their own basal growth by binding to specific receptors expressed on the cell surface.

Animals↗

PubMed1987

Differential purification by immunoaffinity chromatography of two carboxy-terminal portion-deleted derivatives of recombinant human interferon-gamma from Escherichia coli.

Two lower-molecular-weight derivatives of recombinant human interferon-gamma (rIFN-gamma) were purified concurrently from a lysozyme-EDTA extract of Escherichia coli cells by immunoaffinity chromatography using a monoclonal antibody (MAb) against a synthetic carboxy-terminal peptide (Lys-131-Gln-146). The two derivatives, 15K rIFN-gamma and 17K rIFN-gamma, were regarded to have been generated at the extraction step. They were successfully separated from each other by using another MAb against the same synthetic peptide with higher binding affinity than the first. The results of protein-chemical analyses indicate that 15K rIFN-gamma and 17K rIFN-gamma lack 15 (Arg 132-Gln-146) and 4 (Arg-143-Gln-146) carboxy-terminal amino acid residues, respectively. All the data suggest that the two derivatives form a noncovalent dimer and that 15K rIFN-gamma binds indirectly to the MAb column via 17K rIFN-gamma.

Amino Acids↗