Biomedical subjects

M Inomata

Publications and source records attributed to M Inomata.

At least 37 records · Page 2Linked to original sources

PubMed1986

Improved colony formation of cultured retinoblastoma cells.

The effect of various components of three semi-solid media on colony formation in two representative retinoblastoma cell lines, Y-79 and WERI, was determined. Diethylaminoethyl dextran was found to be toxic to the cells, and was deleted from the medium. Horse serum was also used without heat treatment. In the most improved culture medium, plating efficiency was 28% for Y-79 cells and 14% for WERI cells, an increase of more than 10 times that of the original formula. In the new medium, both Y-79 and WERI cells showed relatively constant plating efficiency within a certain range, showing that the medium is useful for quantitative clonogenic study of retinoblastoma cells.

Agar↗

PubMed1985

Detection of NK activity and antibody-dependent cellular cytotoxicity of lymphocytes by human tumor clonogenic assay--its correlation with the 51Cr-release assay.

NK activity of human peripheral blood lymphocytes (PBL) for cells of the human myeloid line K562, and antibody-dependent cellular cytotoxicity (ADCC) of PBL for cells of human lung adenocarcinoma line PC-9 were determined by the human tumor clonogenic assay (HTCA). Incubation of K562 cells or anti-PC-9 serum treated PC-9 cells with PBL before plating inhibited the formation of colonies of these tumor cells. The percent inhibition of tumor cell colony formation was dependent on the effector/target ratio, the incubation time before plating and, in the case of PC-9 cells, on the dilution of anti-PC-9 serum. PBL activated with human T-cell growth factor (TCGF), lymphokine-activated killer (LAK) cells, significantly augmented the inhibition of colony formation of K562 cells, compared to the control lymphocytes. The increase in colony inhibition was dependent on the concentration of TCGF and the time of incubation of PBL with TCGF. The HTCA determining the colony inhibition of K562 cells incubated with LAK or PBL correlated with the 51Cr-release assay (p less than 0.001). The HTCA determining the colony inhibition of anti-PC-9 serum-treated PC-9 cells incubated with PBL also correlated with the 51Cr-release assay (p less than 0.001). We found that the NK activity and ADCC of lymphocytes on K562 and PC9 tumor lines could be detected with HTCA.

Adenocarcinoma↗

PubMed1985

Hydrolysis of protamine by calcium-activated neutral protease (CANP).

To determine the substrate recognition mechanism in calcium-activated neutral protease (CANP), the hydrolytic velocities for some possible substrates were compared. In general, succinylated polypeptides were poorer substrates than unmodified ones, suggesting that CANP interacts with positively charged amino groups and/or repels negatively charged succinyl groups in substrates. Among the substrates examined, protamine was degraded quite rapidly in a restricted manner. This degradation of protamine was remarkably accelerated by the addition of salt, and, in the absence of salt, protamine was inhibitory as to the degradation of vimentin by CANP. Protamine was separated into components and the sites cleaved by CANP were determined. CANP cleaved the clupeine YII and Z components at two sites, both being arginyl-arginine bonds, and the amino acid sequences around these sites were almost identical between YII and Z. No other arginyl-arginine bond was cleaved at all. These results showed that CANP prefers basic amino acid side chains but its specificity is very restricted.

Animals↗

PubMed1985

Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP).

Some endogenous substrates were incubated with two forms of calcium-activated neutral protease (CANP) with high (muCANP) and low (mCANP) sensitivities to calcium ions. In addition to analyses of the processes of their degradation, changes in the molecular properties of these CANPs were also examined. Among the tested substrate proteins, the myosin heavy chain of rabbit skeletal muscle myofibrils and spectrin or band 3 protein of human erythrocyte membranes were degraded relatively rapidly. So far as these proteins were concerned, a higher degradation velocity was observed for muCANP than for mCANP. Vimentin from ascites tumor cells was degraded most rapidly and no difference was observed in degradation velocity between muCANP and mCANP. In all cases, muCANP and mCANP produced different proteolytic peptide fragments, suggesting the different substrate-specificities of these CANPs. The degradation of substrates always accompanied the autodigestion of CANPs, and the small subunits of both CANPs were degraded in the early stage of the autodigestion. The large subunit of muCANP (79K) was converted to a 76K polypeptide via a 77K polypeptide as an intermediate. The autodigested muCANP with 76K polypeptide retained sufficient protease activity and, moreover, its calcium-sensitivity was higher than that of intact muCANP. The possibility is thus proposed that restricted autodigestion is a necessary activation step for the appearance of activity of muCANP. No such transition was observed for mCANP.

Animals↗

PubMed1985

Purification and characterization of 210,000-dalton inhibitor of calcium-activated neutral protease from rabbit skeletal muscle and its relation to 50,000-dalton inhibitor.

An endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle with chemically drastic pretreatments, comprised major (high-molecular-weight form, HMW-inhibitor) and minor (low-molecular-weight form, LMW-inhibitor) components. HMW-inhibitor was purified to homogeneity using FPLC and preparative electrophoresis. The purified inhibitor appeared as a single protein with a molecular weight of 110,000 on SDS-polyacrylamide gel electrophoresis, and a molecular weight of 210,000 on gel filtration. It was therefore presumed that the inhibitor is a dimer protein under native conditions. It contained large amounts of glutamic acid, alanine, and proline, and small amounts of aromatic amino acids, showing an amino acid composition similar to that of LMW-inhibitor. HMW-inhibitor inhibited CANPs with both low (m-type) and high (mu-type) Ca2+-sensitivity but had no effect on any other proteases examined. It was demonstrated that the inhibition was due to the formation of a stoichiometric complex between rabbit mCANP and inhibitor subunit in the ratio of five to one. These results suggest that HMW-inhibitor might have several reactive sites per molecule and that LMW-inhibitor subunit might be a proteolytic fragment of HMW-inhibitor containing an active site.

Amino Acid Sequence↗

PubMed1985