Immunohistochemical demonstration of prostaglandins in various tissues of the rat

To demonstrate the tissue localization of prostaglandin (PG) E₂, PGF₂ and 6-keto-PGF₁ (a stable metabolite of PGI₂) various tissues, including decalcified periodontal tissue of 7-week-old male Wistar strain rats, were immunohistochemically examined using a streptavidin-biotin complex method. Besides tissue macrophages and endothelial cells in various tissues, hepatocytes, renal tubular cells, and parietal and chief cells in the gastric mucosa showed a positive reaction for the various PGs examined. PGs were demonstrated in the cytoplasm or in association with the cell membrane. We generally observed no difference between the localization patterns of PGE₂-, PGF₂-, and 6-keto-PGF₁-positive cells in these tissues. However, in the periodontal ligament and alveolar bone, 6-keto-PGF₁ was localized in the cytoplasm of osteocytes, osteoblasts, cementocytes, and cementoblasts, while no reaction for PGE₂ or PGF₂ was revealed in these cells. We demonstrated the immunohistochemical localization of PGs in various rat tissues including decalcified periodontal tissue and discuss the important roles of PGs in the modulation of their normal functions in these tissues.     

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.     

High-Mr glycoprotein profiles in human milk serum and fat-globule membrane.

In the standard [3H]ouabain-binding assay for quantification of the Na,K-ATPase (Na+ + K+-dependent ATPase) concentration in rat skeletal muscles, samples are incubated for 2 X 60 min in 1 microM-[3H]ouabain at 37 degrees C followed by a wash-out for 4 X 30 min at 0 degree C. To obtain accurate determinations, values determined by this standard assay should be corrected for non-specific uptake and retention of [3H]ouabain (11% overestimation), loss of specifically bound [3H]ouabain during wash-out (21% underestimation), evaporation from muscle samples during weighing (4% overestimation), impurity of [3H]ouabain (5% underestimation) and incomplete saturation of [3H]ouabain binding sites (6% underestimation). Thus corrected the standard [3H]ouabain-binding assay determines the total Na,K-ATPase concentration. Hence, in the soleus muscle of 12-week-old rats the total [3H]ouabain-binding-site concentration is 278 +/- 20 pmol/g wet wt. This is at variance with the evaluation of the Na,K-ATPase concentration from Na,K-ATPase activity measurements in muscle membrane fractions, where the recovery of Na,K-ATPase is only 2-18%. Quantification of the total Na,K-ATPase concentration is of particular importance since it is a prerequisite for the discussion of quantitative aspects of the Na,K-ATPase.     

Effect of aldosterone on dome formation by MDCK mono-layer

Effect of aldosterone on the dome formation in the reconstructed MDCK cell epithelia was studied. MDCK cells derived from dog kidney are assumed to be originated from distal tubules or collecting ducts. When cultured to a confluency, these cells formed a epithelial layer with many domes which contained fluid transported from the apical to the basolateral surface through this layer. Aldosterone at a concentration of 10(-8) to 10(-6) M increased the number of domes dose-dependently, probably through a receptor mediated process, since the dome formation induced by this hormone was completely abolished in the presence of spironolactone. This study primarily disclosed that the dome formation in MDCK cells was stimulated by aldosterone, probably through a receptor mediated mechanism.     

Scale and bone type I collagens of carp (Cyprinus carpio).

1. Soluble Type I collagens were isolated from the scale and bone (skull) of lathyritic carp. Each tissue collagen was assumed to consist of two different molecular forms, (alpha 1)2 alpha 2 as a main component and alpha 1 alpha 2 alpha 3 as a minor one. 2. The possible coexistence of these two forms in soluble Type I collagen of carp was previously observed for skin and muscle, but not for the swim bladder in which only the form of (alpha 1)2 alpha 2 was found. 3. These composite results suggest the wide distribution of alpha 1 alpha 2 alpha 3 heterotrimers in collagenous tissues of carp.     

Mutagenic activity of possible metabolites of 4-nitrobiphenyl ether

A series of possible metabolites--4-nitrosobiphenyl ether (4-NO), 4-hydroxylaminobiphenyl ether (4-NHOH), 4-aminobiphenyl ether (4-NH2), 4-hydroxyacetylaminobiphenyl ether (4-N(OH)Ac), 4-acetoxyacetylaminobiphenyl ether (4-N(OAc)Ac)involved in the toxic effects of 4-nitrobiphenyl ether (4-NO2) was synthesized and tested for mutagenic activity toward Salmonella typhimurium TA100 strain in the presence and the absence of liver homogenates of guinea pig treated with Kaneclor-500. 4-NO2, 4-NO and 4-NHOH showed direct-acting mutagenicity. 4-NO and 4-NHOH showed high mutagenic activity, while the mutagenic activity of 4-NO2 was very weak compared to 4-NO and 4-NHOH. 4-NO showed antimicrobial action at high concentrations. The other three compounds tested induced no mutation. Upon addition of NAD(P)H, the mutagenic activities of 4-NO and 4-NHOH were slightly enhanced, but no enhancement was observed by addition of NAD(P)+. Metabolic activation with guinea pig liver homogenates enhanced the mutagenic activities of 4-NO2 and 4-NO, and converted 4-NH2, 4-N(OH)Ac and 4-N(OAc)Ac to the product(s) responsible for the mutagenic activity. Addition of bis(p-nitrophenyl)phosphate, a deacetylase inhibitor, inhibited the mutagenic activities of 4-N(OH)Ac and 4-N(OAc)Ac by about 70% in the presence of NADPH and about 77% in the absence of NADPH. High performance liquid chromatography (HPLC) analysis of non-enzymatic conversion-products of 4-NHOH and 4-BO with and without NADPH indicated that 4-NHOH disappeared after 30 min of incubation and was converted completely to 4-NO without NADPH, while with NADPH, 4-NHOH disappeared very slowly and was detected even after 4 h of incubation. In the case of 4-NO, no decrease of 4-NO was observed without NADPH, while with NADPH 4-NO decreased quickly and a significant amount of 4-NHOH appeared. The mechanism of the NAD(P)H-dependent increase in mutagenicity is also discussed.     

Sex steroid-binding protein in a subline of Dunning R 3327 prostatic adenocarcinoma of the rat

A transplantable tumor CUB-II, a subline derived from the Dunning R 3327 rat prostatic adenocarcinoma, contains a unique sex steroid-binding protein. The protein possesses binding sites for androgens as well as for estrogens, and the binding affinity to androgen is higher than that to estrogen. The sedimentation coefficient of the protein is 10S. Sodium thiocyanate inhibits the binding to both sex steroids. This type of binding is not present in the 0.4M KC1 extract of nuclei. These results suggest that the binding protein is not the receptor for steroid hormones in spite of its high affinity binding to androgens and estrogens. Since the original tumor does not contain such protein, production of this binding protein seems to take place during culture in vitro and/or serial transplantations of the tumor.     

Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography

A rapid and simple analytical method for unsaturated disaccharide isomers formed by enzymatic digestion from hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin by high-performance liquid chromatography using an amine-bound silica column with a linear gradient of sodium dihydrogen phosphate was developed. The analyses were performed on isomers of two groups belonging to the chondroitin sulfate family and the heparin sulfate family. In both families, disaccharide isomers eluted in the order non-, mono-, di-, and trisulfated disaccharides by elevating salt concentrations. The method was applied to the analysis of constituent disaccharides of representative sulfated glycosaminoglycans, which proved that most constituents could be quantified separately. This method is advantageous in that enzymatic digests can be applied directly on a column without any pretreatment and good resolution of several disaccharides can be obtained by one chromatography.     

Study on the interaction of aromatic dyes with nucleic acids by means of UV, CD and NMR spectroscopies.

The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies.