Enzymology and Genetics of Halogenating Enzymes from Bacteria

This paper discusses the properties of bacterial haem-containing and non-haem haloperoxidases, their involvement in the biosynthesis of halometabolites and their use in bioconversion. The very low peroxidase activity of bacterial non-haem haloperoxidases, their stability at high temperature and over a wide pH-range makes them particularly suited for use in the bromination of organic compounds. The chloroperoxidase from Pseudomonas pyrrocinia is the only haloperoxidase showing substrate specificity and regioselectivity. The genes of one chloro- and one bromoperoxidase could be cloned. The corresponding enzymes can now be produced in large amounts and at low costs.     

Change in floral nectar components from fresh to senescent flowers ofCapparis spinosa (Capparidaceae), a nocturnally flowering Mediterranean shrub

We studied the nectar characteristics in relation to flower age of the summer flowering Mediterranean shrubCapparis spinosa in three localities in Southern Greece. Anthesis was nocturnal. Nectar volume, concentration, and sucrose/hexose ratio varied with site, year, and between individual plants; amino acid concentration varied only with site. The sucrose/hexose ratio decreased considerably with flower age, while the glucose/fructose ratio remained constant (ca. 1), implying that nectar sucrose broke down in the course of anthesis. Sugar breakdown increased with water content of nectar. Amino acid concentration was strongly age-dependent: It was low in fresh flowers, relatively high in middle-aged ones (except aspartic acid that was extremely increased), and very high in senescent ones. We attribute the amino acid changes to phenomena related to flower senescence in the dark.     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

Analysis of Cytokinin Metabolism in ipt Transgenic Tobacco by Liquid Chromatography-Tandem Mass Spectrometry

The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3H]isopentenyladenine and [3H]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokininoxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.     

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.