Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression

The nucleotide and amino acid sequences for rat type I angiotensin II receptor were deduced through molecular cloning and sequence analysis of its complementary DNAs. The rat angiotensin II receptor consists of 359 amino acid residues and has a sequence similar to G protein-coupled receptors. The expression of this receptor gene was detected in the adrenal, liver and kidney by Northern blotting. Sodium deprivation positively modulated the expression of the receptor gene in the adrenal. No detectable change was observed in the expression levels of this receptor gene between spontaneously hypertensive rats and Wistar-Kyoto rats in the tissues examined including the adrenal, brain, kidney and liver. Interestingly the expression of this receptor gene was developmentally regulated.     

Analysis of non-heme iron in arachidonate 12-lipoxygenase of porcine leukocytes

Arachidonate 12-lipoxygenase of porcine leukocytes, which was purified to homogeneity by immunoaffinity chromatography, was analyzed for iron content by atomic absorption spectrophotometry. The enzyme contained 0.70 +/- 0.09 g atom of iron per mol of enzyme (mean +/- S.D., n = 4). Inorganic iron, which was added to the enzyme solution as an internal standard, was recovered in almost 100% yield. Among various iron chelators tested, only 2,2'-dipyridyl at 1 mM inactivated the enzyme by 87%, but the enzyme was not reactivated by the addition of excess ferrous or ferric iron.     

Establishment and characterization of new cell lines from human renal cell carcinoma

We have characterized seven human renal cell carcinoma cell lines established from primary sites of five patients between 1987 and 1989. Two lines, OUR-20P and OUR-20S, were derived from the OUR-20 cells by cloning with a dilution method 3 months after the primary culture. These three cell lines were tumorigenic in athymic nude mice when inoculated subcutaneously. Examined by a dye uptake method, OUR-20 was highly sensitive to interferon-alpha (IFN-alpha); OUR-20P, OUR-20S and OUR-30 showed slight sensitivities, while the other three cell lines were insensitive. All seven cell lines have been maintained for more than 2 years and over 50 passages in vitro. Cytogenetic analyses performed 1.5 to 3 years after the starts of primary cultures indicated that all seven cell lines, which exhibited different morphologies in phase-contrast micrographs, were aneuploid with modal chromosome numbers 41 to 89.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Purification and characterization of soluble human IL-6 receptor expressed in CHO cells

An immunosorbent assay system to detect genetically engineered IL-6 receptor (IL-6R) was established, whereby soluble IL-6 receptor (sIL-6R) was detected in the culture medium when sIL-6R cDNA was transfected into COS1 cells. A stably transformed Chinese hamster ovary (CHO) cell line constitutively expressing sIL-6R has been established. The recombinant sIL-6R was purified to homogeneity by sequential filtration and chromatography of the culture medium. The recombinant sIL-6R augmented the sensitivity of M1 cells to IL-6 in growth inhibition assay in a dose-dependent manner. Furthermore, a radioisotope immunosorbent assay (RIA) utilizing recombinant sIL-6R was established which could detect IL-6 in a quantity as low as 10 ng/ml.     

Hydrophobic residues 382-386 of antithrombin III, Ala-Ala-Ala-Ser-Thr, serve as the epitope for an antibody which facilitates hydrolysis of the inhibitor by thrombin.

In the presence of a monoclonal antibody raised against the human thrombin-antithrombin III complex, the reaction between thrombin and antithrombin III proceeded to form preferentially a two-chain form of the inhibitor rather than to follow the major pathway of stable acyl complex formation. We thus propose the term "switching antibody" for an antibody that switches the enzyme-inhibitor reaction (Asakura, S., Matsuda, M., Yoshida, N., Terukina, S., and Kihara, H. (1989) J. Biol. Chem. 264, 13736-13739). By analyzing a CNBr fragment of the thrombin-antithrombin III complex that reacts with the antibody we localized the epitope for the antibody to a strongly hydrophobic residue 382-386 peptide segment, Ala-Ala-Ala-Ser-Thr, of the inhibitor, which is also contiguous with a hydrophobic amino acid Ala at its carboxyl terminus. This particular region should be cryptic in nascent antithrombin III, but could have been exposed to provide the reactive site for the antibody at an early stage of the reaction. Thereby a conformational change may have been induced at or near the reactive site of the complex, facilitating hydrolysis of the inhibitor by the enzyme. Interestingly, this hydrophobic region is highly conserved among members of the serpin family.     

Productivity and mineral cycling in an oak coppice forest 2. Annual net production of the forest

Annual net production was estimated in the secondary coppice forest near Tokyo, which was dominated by a deciduous oak,Quercus serrata Thunb. Lateral growth of stems and old branches was directly estimated by examining the annual rings for 35 shoots in a clear-cut quadrat of 10m×10m. Phytomasses of current organs were also weighed in the quadrat. Preharvest losses of current organs were determined by twelve 0.5 m² litter traps for fine litter and twelve 6 m² quadrats for woody litter. Branch production was also assessed indirectly by use of the stem-branch allometry and death of branches. The results of the indirect method were in sufficient agreement with the result of the direct one. Grazing loss of leaves from the canopy was estimated directly from the loss in leaf area and indirectly from the animal faeces caught by the litter traps. Net production of the canopy trees was 149 kg a⁻¹ year⁻¹, in which leaf production was 36.9 kg. Animals grazed about 14% of the leaf area by the end of the growing season. True consumption of leaves by animals was 7.6% of leaf production or 10% of leaf mass. Production of undergrowth, mainly a dwarf bamboo,Pleioblastus chino Makino, was 28 kg a⁻¹ year⁻¹, being 15% of the total stand production. Productivity of this forest was significantly higher than that of cool-temperate deciduous broadleaf forests.     

Biosynthesis of xyloglucan in suspension-cultured soybean cells. Occurrence and some properties of xyloglucan 4-beta-D-glucosyltransferase and 6-alpha-D-xylosyltransferase

A particulate enzyme fraction that catalyzes the transfer of glucose from UDP-[14C]glucose and of xylose from UDP-[14C]xylose into a xyloglucan has been isolated from suspension-cultured soybean cells. The incorporation of radioactivity from [14C]xylose into the polysaccharide was dependent on the presence of UDP-glucose in the incubation mixture, and that from [14C]glucose was dependent on the concentration of UDP-xylose in the mixture. Mn2+ was required for the incorporation of xylose and the optimum concentration of Mn2+ was about 10 mM. This reaction showed a pH optimum at 6.5 to 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and was inhibited by phosphate buffer and Tris buffer. On hydrolysis with Trichoderma endoglucanase, the polysaccharide synthesized in vitro gave a pentasaccharide, a hepatasaccharide, and a small amount of non-asaccharide. Based on the results from fragmentation and methylation analyses, the following structures were proposed for the penta- and the heptasaccharides from the xyloglucan synthesized in vitro: (formula, see text).     

Biosynthesis of Xyloglucan in Suspension-Cultured Soybean Cells. Synthesis of Xyloglucan from UDP-Glucose and UDP-Xylose in the Cell-Free System

When UDP-[¹⁴C]glucose or UDP-[¹⁴C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave ¹⁴C-monosaccharides and a ¹⁴C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981)     

Biosynthesis of Xyloglucan in Suspension-cultured Soybean Cells. Evidence that the Enzyme System of Xyloglucan Synthesis does not Contain {beta}-1,4-Glucan 4-{beta}-D-Glucosyltransferase Activity (EC 2.4.1.12)

Xyloglucan 4-ß-D-glucosyltransferase, an enzyme responsiblefor the formation of the xyloglucan backbone, in a particulatepreparation of soybean cells has been compared with ß-1,4-glucan4-ß-D-glucosyltransferase of the same origin. Thefollowing observations indicate that the enzyme system of xyloglucansynthesis does not contain ß-1,4-glucan 4-ß-D-glucosyltransferaseactivity, although both enzymes transfer the glucosyl residuefrom UDP-glucose to form the ß-1,4-glucosidic linkage:1. The incorporation of [¹⁴C]glucose into xyloglucan dependedon the presence of UDP-xylose in the incubation mixture. 2.No measurable amount of radioactivity was incorporated fromUDP-[¹⁴C]xylose into the cello-oligosaccharides, although theincorporation of [¹⁴C]xylose into xyloglucan depended on thepresence of UDP-glucose in the incubation mixture (Hayashi andMatsuda 1981b). 3. The activity of xyloglucan 4-ß-D-glucosyltransferasewas stimulated more strongly by Mn²⁺ than by Mg²⁺, whereas Mg²⁺was the most active stimulator for the activity of ß-1,4-glucan4-ß-D-glucosyltransferase. 4. An addition of GDP-glucose(100 µM) to the incubation mixture inhibited the activityof xyloglucan 4-ß-D-glucosyltransferase by 17%, whereasthe activity of ß-1,4-glucan 4-ß-D-glucosyltransferasewas inhibited 56% under the same conditions. 5. Irpex exo-cellulasedid not hydrolyze the xyloglucan synthesized in vitro. 6. Theß-1,4-glucan synthesized in vitro was not a branchedxyloglucan because it gave no 2,3-di-O-methyl glucose derivativeon methylation analysis. 7. Pulse-chase experiments indicatedthat the ß-1,4-glucan was not transformed into thexyloglucan. The subcellular distribution of the xyloglucan synthase, however,was similar to that of the ß-1,4-glucan synthase (Golgi-located1,4-ß-D-glucan 4-ß-D-glucosyltransferase).Thus, it appears that the latter enzyme is located at a siteclose to xyloglucan synthase and is set aside for the assemblyof these polysaccharides into the plant cell surface. (Received May 21, 1981; Accepted October 13, 1981)