Takashi Kamogashira

Site-specific mutagenesis of the human interleukin-1β gene: Structure-function analysis of the cysteine residues

Human interleukin-1 beta (IL-1 beta) has two cysteines located at amino acid residues 8 and 71 of the mature protein consisting 153 amino acids. To clarify the role of these characteristic cysteine residues in IL-1 beta, at first, an expression plasmid for site-specific mutagenesis has been constructed by inserting the ori and intergenic region of phage f1 into the IL-1 beta expression vector. The plasmid can be used not only for isolation of the modified IL-1 beta gene but for expression of the mutant protein in Escherichia coli. Using this plasmid, each of the cysteine codons in IL-1 beta gene was changed to serine or alanine codon, or deleted. The modified IL-1 beta showed that the two cysteine residues in IL-1 beta are not essential for biological activity but not to be eliminated for the maintenance of the functional structure of IL-1 beta.

Amino-terminal region of human macrophage colony-stimulating factor (M-CSF) is sufficient for its invitro biological activity: Molecular cloning and expression of carboxyl-terminal deletion mutants of human M-CSF

Human T lymphoblastoid cell line CEM-ON belongs to a helper/inducer subclass and secretes M-CSF into medium constitutively. We have isolated a full-length cDNA clone for this factor from a cDNA library of this cell line. The cDNA was 2.5 kb and coded for a 554 amino acid polypeptide precursor including signal sequence. The Northern blot analysis showed that the major transcript of M-CSF is about 4.2 kb. We have studied the expression of not only the wild type plasmid, but also the five C-terminal deletion mutants encoding N-terminal 154, 163, 170, 177, and 185 amino acid residues in monkey COS-1 cells. The results showed that at least C-terminal 377 amino acid residues of human M-CSF are not essential to manifest the in vitro biological activity on murine bone marrow cells.

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Isolation of a fosfomycin-hypersensitive mutant and production of α-d-glucose-l-phosphate from Bacillus sp. BA-3796 screened by its use

Abstract The development of a very sensitive and highly specific screening method for detection of new cell wall inhibitors of the fosfomycin type is described. A fosfomycin-hypersensitive mutant, f-360, was isolated from Staphylococcus aureus Newman by selection with fosfomycin, an antibiotic that inhibits synthesis of the bacterial cell wall. The mutant f-360 was 50-fold more sensitive than the parent strain to fosfomycin. The mutant was constitutive for the hexose phosphate transport system. Using the organism in screening, BA-3796, which had an antibacterial activity against mutant f-360 was found to be produced by a bacterium designated Bacillus sp. BA-3796. Starch and beef extract were the most suitable carbon and nitrogen sources for BA-3796 production and the amount of BA-3796 reached 3 g/ l at a maximum level. The purified BA-3796 was identified as α- d -glucose-l-phosphate by its various physiochemical properties. α- d -Glucose-1-phosphate showes an antibacterial activity against Staphylococci in the presence of a slight amount pf α- d -glucose-6-phosphate.

Effects of media conditioned by a non-metastasizing human salivary gland adenocarcinoma cell clone and metastasizing clones from salivary gland and various other tissues on the proliferation, migration and protease production of bovine aortic endothelial cells in vitro

We demonstrate the role in tumor-associated angiogenesis of factors released into conditioned medium (CM) from in vitro human salivary gland cell clones with biological phenotypes ranging from non-metastasizing to metastasizing. A non-metastasizing human salivary gland adenocarcinoma cell clone HSGc and its subclone with metastatic potential (Gc2-100 cl-1) were employed. We also used metastasizing cell clones obtained by explant cultures of organs of Gc2-100 cl-1 tumor-bearing nude mouse. The proliferation and migration of bovine aortic endothelial (BAE) cells were significantly stimulated by the addition of CM obtained from Gc2-100 cl-1 and metastasizing cell clones, while CM from HSGc was ineffective. When the effect on protease secretion by BAE cells was examined, CM from Gc2-100 cl-1 and metastasizing cell clones inhibited the secretion of type IV collagenases by BAE cells much more than did CM from HSGc. These findings, therefore, may imply that Gc2-100 cl-1 and metastasizing cell clones secrete angiogenic factors that stimulate not only the proliferation and migration of endothelial cells but also the formation of basement membrane components necessary for the reconstruction of new blood vessels at migrated sites of endothelial cells by preventing the degradation of basement membrane component, type IV collagen.