Simultaneous Determination of Oxidized and Reduced Forms of Plastoquinone A in Spinach Chloroplasts by High-Performance Liquid Chromatography with an Electrochemical Detector

A method for determination of the redox level of plastoquinoneA in spinach chloroplasts is described. Plastoquinone A andits reduced form plastoquinol A were extracted from chloroplastson a sample-preparation cartridge (SEP-PAK C₁₈ Cartridge, WatersAssoc. Inc.) with a mixture of ethanol and diethyl ether ( 1: 1, vv). Extracts were separated by reversed-phase high-performanceliquid chromatography and examined with an electrochemical detectorequipped with dual electrodes. Plastoquinone A was determinedby its reductive current on one electrode, and plastoquinolA by its oxidative current on the other electrode. This method was applied to the determination of the redox potentialof plastoquinone A in chloroplasts. The midpoint potential atpH 7.8 of plastoquinone A was +20 mV with an n number of 2. (Received March 30, 1987; Accepted August 3, 1987)     

High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe.

We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 10(6) colonies per 10(8) cells transfected with 2 micrograms of library and 1 microgram of vector, 50-60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz. pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast. This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.     

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.     

Sulfated glycoproteins synthesized by the corneal epithelium of chick embryo having the same molecular weights as cytokeratin polypeptides

The previous study from this laboratory demonstrated that the corneal epithelium of 19-d-old chick embryo synthesizes two classes of sulfated glycoconjugates consisting of sulfated glycoproteins and proteoglycans (Yonekura, H., Oguri, K., Nakazawa, K., Shimizu, S., Nakanishi, Y., & Okayama, M. (1982) J. Biol. Chem. 257, 11166-11175). The present study demonstrated that when the sulfated glycoproteins labeled metabolically with [35S]sulfate and [3H]glucosamine were analyzed by SDS-PAGE, the 70,000 component (accounting for approximately 30% of the 35S and 35% of the 3H of the total sulfated glycoprotein) co-migrated with five major proteins with apparent molecular weights (Mrs) of 70,000, 66,000, 58,000, 51,000, and 48,000, which together accounted for about 57% of the total tissue protein. All five proteins cross-reacted with an antibody against human sole keratin, indicating that they are cytokeratin polypeptides of the corneal epithelium. Amino acid analysis demonstrated that they had high contents of glycine, serine, glutamic acid, leucine, and aspartic acid. Two-dimensional tryptic peptide maps indicated that they were all different. Analysis of radiolabeled materials released by alkaline borohydride treatment of the sulfated glycoproteins which were synthesized in the presence and absence of tunicamycin and co-purified with the five cytokeratin polypeptides, revealed that they contained both N- and O-glycosidically linked sulfated oligosaccharides. All the results obtained in the present study indicate that the five sulfated glycoproteins are similar, if not identical, to the cytokeratin polypeptides. This is consistent with the result in the accompanying paper that these sulfated glycoproteins are localized intracellularly.     

Plasmid R46 provides a function that promotes recA-independent deletion, fusion and resolution of replicon

Summary We report that plasmid R46 provides a function which promotes recA-independent deletion, replicon fusion, and resolution of the fusion. R46 belongs to the incompatibility group N and specifies resistance to ampicillin, tetracycline, streptomycin and sulfonamide. Four kinds of deletion derivatives were observed by selection for susceptability to tetracycline from ampicillin-resistant clones. A common region, will be called region thereafter, was postulated to be involved in these deletions. The replicon fusion occurred by a conjugative mobilization of each derivative with plasmid R388. The fusion was suggested to contain both replicons linked at each junction by the sequence in the region in direct orientation. The resolution of the replicon fusion was found between two regions and a consequently generated, parental deletion derivative and an R388 derivative which gained one region. It is possible that the region contains one potential Insertion Sequence (IS) element. These events were also speculated to occur as a consequence of insertion of the potential IS onto the intramolecular or intermolecular target sequence, or reciprocal recombination between two potential IS elements.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Urocystis tranzscheliana,a newly recorded smut fungus onPrimula sieboldii from Japan

A smut fungus onPrimula sieboldii was newly found in Japan and identified asUrocystis tranzscheliana by comparative morphology. This species causes systemic infection ofP. sieboldii and produces sori in its ovaries.Contribution No. 121, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.