A new enzyme, NADPH-dihydropteridine reductase in bovine liver.

An enzyme designated as NADPH-dihydropteridine reductase was found in the extract of bovine liver and partially purified. In contrast to NADH-dpendent dihydropteridine reductase [EC 1.6.99.7], the enzyme catalyzes the reduction of quinonid-dihydropterin to tetrahydropterin in the presence of NADPH. The two enzymes were separated by column chromatography on DEAE-sephadex. Tyrosine formation in the phenylalanine hydroxylation system was also stimulated by NADPH-dihydropteridine reductase. The existence of these two dihydropteridine reductases suggests that the tetrahydro from ofpteridine cofactor may be regenerated in two different ways in vivo.     

Studies on fish scale formation and resorption

Summary Electron microscopic investigation of scales of the goldfish Carassius auratus revealed that the lamellae of fibrillary plates contain sheet-like structures composed of vertically oriented collagen fibers embedded in an organic matrix. The fibers (TC fibers) are smaller in diameter (35–45 nm) than those of the lamellae and the matrix is stained intensely with lead citrate.The sheet-like structures as well as the lamellae are formed by fibroblasts located beneath the lamellae. The orientation of the collagen fibers of the sheets and the lamellae seems to be controlled by the orientation of the ridges and invaginations of the surface of the fibroblasts.The fibrillary plate of C. auratus was found to be partially calcified. Calcification was initiated by the deposition of needle-like or flaky crystals of hydroxyapatite in the organic matrix of the sheet-like structure and proceeded into the TC fibers and the matrix region of the lamellae. The potassium pyroantimonate-osmium tetroxide method showed a heavy concentration of calcium in the osteoblasts, fibroblasts, and in the matrix regions of the fibrillary plate. Calcium-containing precipitates were also present in the hole zone of the collagen fibers in the lamellae, but the significance of this location in calcification remains to be elucidated.Contribution No. 285, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

Effect of suramin on growth of androgen-responsive mouse tumor (Shionogi carcinoma 115) and its autonomous subline (Chiba subline 2)

Suramin has been shown to inhibit the binding of various growth factors to their receptors. Shionogi Carcinoma 115 cells (SC 115 cells) and Chiba Subline 2 cells (CS 2 cells) are clones of an androgen-responsive mouse tumor cell and its autonomous subline, respectively. Since the growth of SC 115 and CS 2 cells are assumed to be regulated by their own fibroblast growth factor (FGF)-like growth factors, the present study was undertaken to examine the effect of suramin on these cells. Suramin inhibited the growth of SC 115 and CS 2 cells in a dose dependent manner. The inhibition of suramin was reversible up to 50 micrograms/ml. Suramin reversibly changed the shape of these cells from fibroblast-like to polygonal and epithelial-like ones, and inhibited 3H-thymidine incorporation into these cells which was evoked by acidic and basic FGFs, and conditioned medium obtained from CS 2 cells. The binding of 125I-basic FGF to SC 115 and CS 2 cells was inhibited by suramin. However, suramin had no effect on growth factor production and the hst-1 gene expression on CS 2 cells. In conclusion, suramin inhibited the autocrine and paracrine growth of SC 115 and CS 2 cells by blocking the binding of autocrine growth factors to their receptors.     

A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk

When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM. From the site-directed mutagenesis of SpG and PPP1R5, it was clarified that (i) the association rate constant of the Lys454Pro/Glu456Gln mutant of SpG to goat immunoglobulin G was almost equivalent to that of the wild-type but its dissociation rate constant was about 2.7 times that of the wild-type and (ii) the amino acid substitutions of Phe180 in PPP1R5 reduced glycogen-binding by 30-50%. Since HAFCOM using the GFP-tagged analyte requires no special chemicals and instruments, this system can easily and economically assay the specific interaction between target protein and ligand.     

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.