Sachiya Ohtaki

Cloning and characterization of rat p27Kip1, a cyclin-dependent kinase inhibitor

Cyclin-dependent kinase (Cdk) inhibitors play significant roles in the cell cycle control of various biological phenomena. To characterize the role of Cdk inhibitors in rat cells, we isolated a cDNA encoding rat p27Kip1, a 27-kDa Cdk inhibitor. The 1.04-kb cDNA of rat p27 contained an open reading frame of 197 amino acids that shared high homology with mammalian p27 and significant homology with mammalian p21Cip1 and p57Kip2. p27 mRNA was detected in most rat tissues and cell lines. The levels of p27 protein expression were similar in rat cell lines transformed by E1A and in normal cells. Rat p27 was able to interact with Cdk 2/4 and cyclin A/D in rat cells, but the amounts of rat p27 in Cdk2 complexes were different between transformed cells and normal cells. Thus, the formation of stable complexes of rat p27 may be modulated by E1A. Rat p27 protein could inhibit the increased Cdk2-associated kinase activity in transformed rat cells.

A sandwich enzyme immunoassay of human growth hormone in serum using anti-human growth hormone IgG-β-D-galactosidase conjugate

A sandwich enzyme immunoassay for the measurement of human growth hormone in serum was developed using antibody IgG-beta-D-galactosidase conjugate and antibody IgG-coated silicone rubber rods. The sensitivity of the assay was 4 pg/tube, which corresponded to 0.4 ng/ml, when 10 microliters of serum was used. The assay system showed little cross-reaction to other human pituitary hormones including prolactin, thyroid-stimulating, follicle-stimulating and luteinizing hormones and human chorionic gonadotropin. The coefficients of variation for different levels of serum hGH ranged from 5.8-9.3% and 6.7-14.5%, for within and between run, respectively. The regression equation and coefficient for correlation to radioimmunoassay were gamma (EIA) = 1.06 kappa (RIA) -0.22 and 0.97 (n = 46), respectively.

Hydrogen peroxide generating system in hog thyroid microsomes

Abstract 1. 1. The NADH oxidation caused by hog thyroid microsomes was studied. Time courses of the oxidation of NADH and the reduction and oxidation of microsomal cytochrome b5 were followed simultaneously by the use of a dual-wavelength spectrophotometer. 2. 2. The turnover number of the bound cytochrome b5 in the NADH oxidation was calculated to be 0.13 s−1 according to Chance. From the kinetic trace of the cytochrome b5 oxidation the rate of the oxidation was calculated to be 0.037 s−1. The discrepancy between these two values was discussed. 3. 3. The H2O2 generation was confirmed by use of hydroperoxidase inhibitors and cytochrome peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5). By the latter method, the amount of H2O2 generation was determined to be 40% of the amount of NADH oxidized.

Cloning and characterization of a novel RNA-binding protein SRL300 with RS domains.

AT-rich element binding factor 1 (ATBF1) mRNA encodes a transcription factor implicated in neuronal differentiation. A cDNA for the protein that can bind the 5-noncoding sequence of the ATBF1 mRNA was cloned. The deduced protein, termed SRL300, contains a unique RNA-binding region, two large RS domains and many phosphorylation sites. SRL300 protein was detected in both human and rat cells.

Reaction of detergent-solubilized thyroid peroxidase with hydrogen peroxide

Thyroid peroxidase is a membrane-bound enzyme that catalyzes the biosynthesis of thyroid hormones. The enzyme has been partially purified after solubilization by proteolysis, mostly combined with detergent treatment [l-l I]. Spectral properties have been reported on such enzyme preparations but little on the enzyme solubilized by detergent treatment only. Although thyroid peroxidase is believed to react with hydrogen peroxide to form catalytic intermediates such as compounds I and II, their existence has not yet been detected by spectrophotometric methods. Here we report some spectral properties of detergent-solubilized hog thyroid peroxidase and show spectrophotometric evidence which indicates the formation of compound II of the enzyme.

An Enzyme-Linked Immunoassay for the Measurement of Circulating Immune Complexes

A solid-phase, enzyme-linked immunoassay for the quantitation of circulating immune complexes was developed using C1q. In this assay, the immune complexes in human serum are adhered to C1q-coated polystyrene tubes and then detected by a β-D-galactosidase-conjugated monovalent fragment (Fab′) of rabbit (anti-human IgG, γ chain-specific) IgG. The enzyme activity bound to the tubes was determined fluorometrically using 4-methylumbelliferyl-β-D-galactopyranoside as substrate. The sensitivity of this assay was as little as 100 ng/tube, which corresponded to 6 μg/ml when 16·7 μl of serum was assayed. The specificity of the assay was demonstrated by the following observations: (1) absence of cross-reaction of monomeric IgG; (2) non-detectability of immune complex in most of the sera from normal subjects; (3) parallelism of the standard curve with dilution of reference serum. The precision of the assay was proved by the demonstration of sufficient within-assay coefficients of variation (10·7–14·9%).

A Simultaneous Enzyme-Linked Immunoassay for Anti-Thyroglobulin Autoantibody in Human Serum

Abstract A “simultaneous” enzyme-linked immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum was studied, in which human thyroglobulin-coated silicone rubber rod, s-D-galactosidase- or horseradish peroxidase-conjugated human thyroglobulin and serum sample were mixed at the same time. In order to obtain a suitable range of the measurable antibody, an appropriate amount of human thyroglobulin-enzyme conjugate was carefully selected. A successful simultaneous assay for antithyroglobulin was obtained using thyroglobulin-s-D-galactosidase conjugate, in which the minimum amount of detectable antibody was approximately 10 ng/ml using 5 μl of serum. This sensitivity was 20-fold higher than that in two-step sandwich enzyme-linked immunoassay reported previously.