ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.     

Determination of imidazobenzodiazepine-3-car☐amide, a new anxiolytic agent, in human plasma by gas chromatography—negative chemical-ionization mass spectrometry

A method is described for measuring imidazobenzodiazepine-3-car☐yamide, a new anxiolytic agent, in human plasma. A tetradeuterated analogue of the analyte is used as the internal standard. The drug and its internal standard are (1) extracted from plasma at pH 9 with benzene containing 20% 1, 2-dichloroethane, (2) derivatized with pentafluoropropionic anhydride in the presence of triethylamine and (3) the nitrile derivative of the analyte and internal standard are analyzed by gas chromatography (GC)—negative chemical-ionization mass spectrometry (CIMS) using methane as both GC carrier gas and CI reagent gas. The mass spectrometer is set to monitor the intense (M-HCl)⁻- ions of imidazobenzodiazepine-3-nitrile and its tetradeuterated analogue atm/z 316 andm/z 320, respectively. Quantitation of an experimental plasma sample is based on the comparison of them/z 316 tom/z 320 ion ratio in each sample to that obtained from the analyses of control plasma spiked with various amounts of the drug and a fixed amount of internal standard. The limit of quantitation of the method is approximately 100 pg ml⁻¹ of plasma and the precision (relative standard deviation) at a plasma concentration of 1 ng ml⁻¹ is 4%.     

Coculture of Astroglial and Vascular Endothelial Cells as Apposing Layers Enhances the Transcellular Transport of Hypoxanthine

Abstract: In brain, astrocytes and endothelial cells are a major site of adenosine degradation. These two cell types, found in close apposition, constitute the wall of the brain's capillaries and serve as a site of hypoxanthine production and degradation. Both cell types possess the hypoxanthine salvage pathway and can incorporate hypoxanthine into nucleotides. This suggests that the endothelial-astrocyte anatomical complex might play an important role in the brain's purine homeostasis. To test this hypothesis, cocultures of monolayers of vascular endothelial cells and astrocytes were grown over a porous membrane, in close apposition to one another, and studies on hypoxanthine transport and metabolism to uric acid were performed. The flux of hypoxanthine across the cell layers was simultaneously determined and compared with the flux of sucrose, as a probe of passive diffusion. Our results show that in endothelial, glial, and endothelial-glial cell layers the hypoxanthine flux was greater than that of sucrose, and that the flux of hypoxanthine, but not of sucrose, was inhibited by adenine or by lowering the temperature. These results suggest that hypoxanthine moves across endothelial, glial, and endothelial-glial cell layers by a transport process. Furthermore, we found that hypoxanthine transport is enhanced when glial and endothelial cells are cocultured compared with that in glial or endothelial monolayers. In addition the coculture also resulted in a depression of xanthine oxidase activity.     

Increased incidence of disomic sperm nuclei in a 47,XYY male assessed by fluorescent in situ hybridization (FISH)

Using triple-colour fluorescent in situ hybridization in decondensed sperm heads, we assessed the sex-chromosome distribution in spermatozoa from a 47,XYY male compared with controls. The incidence of spermatozoa with 24,XY (0.30%) and 24,YY (1.01%) disomy was significantly higher than in our control series. Diploid meiocytes present in the ejaculate were mainly 47,XYY (60.6–86.7%), and haploid meiocytes were mainly 24,XY (78.1%).These results suggest that, although the extra Y chromosome is thought to be eliminated during spermatogenesis, XYY germ cells can complete meiosis and produce disomic spermatozoa. Received: 5 August 1996 / Revised: 2 October 1996     

Transformation of Penicillium chrysogenum to sulfonamide resistance

Penicillium chrysogenum has been transformed to sulfonamide resistance by vectors containing the dihydropteroate synthetase gene from plasmid R388 controlled by the promoter and terminator sequences of the P. chrysogenum trpC gene. Transformation frequencies of four to ten transformants per microgram of vector DNA were obtained.     

Characteristics of preleukemia cells induced in mice.

A high proportion of irradiated C57BL/6 mice inoculated with the radiation leukemia virus D-RadLV develop overt T-cell leukemias originating in the thymus. In unirradiated hosts the incidence is much lower. As early as 10 days after injection of D-RadLV the bone marrow contains “preleukemia” cells which, although not frankly leukemic, will develop into leukemia cells if transferred into a specially pretreated recipient mouse. In the present report, certain properties of D-RadLV-induced leukemia and preleukemia cells are compared. In this model system, leukemia cells express the T-cell surface component Thy-1 (Thy-1⁺) whereas preleukemia cells do not (Thy-1^?). But preleukemia cells could be induced in vitro by thymopoietin or ubiquitin to become Thy-1⁺, suggesting that they are prothymocytes. Unlike leukemia cells, preleukemia cells injected into normal recipients immunized them against transplants of leukemias induced by the same D-RadLV virus. Evidently D-RadLV virus induces a critical change in prothymocytes which in a later (Thy-1⁺) phase of differentiation is manifest in overt leukemia transformation.     

Ecophysiology of six selected shrub species in different plant communities at the periphery of the Atlantic Forest of SE-Brazil

The Atlantic rain forest of Brazil is one of the 25 biodiversity hotspots of the world. It is surrounded by distinct marginal plant communities which are floristically related to the rain forest. We performed an in situ ecophysiological study of six shrub and tree species which are abundant in one or more of these marginal ecosystems. They formed three pairs of taxonomically related species, which, however, differed in geographic distribution and/or habitat preference. We aimed to answer the following questions: (1) Do habitat generalists and specialists differ in ecophysiological behaviour? (2) Are there general trends responsive to site characteristics, irrespective of taxonomic affiliation? (3) In which variables between-site intraspecific variation is more often detected? Plants chosen were the Clusiaceae Calophyllum brasiliense Cambess (swamp forest specialist) and Rheedia brasiliensis (Mart.) Planch &; Triana (a generalist plant, occurring in swamp forests and dry, sandy coastal vegetation); the Myrsinaceae Myrsine parvifolia A.DC. (widespread in sandy plains) and Myrsine gardneriana A.DC. (widespread in high-altitude habitats); and the Euphorbiaceae Styllingia dichotoma Muell. Arg. (a coastal inselberg specialist) and Croton compressus Lam. (a generalist found in coastal inselbergs and dry forests). We compared measurements of photosynthesis (chlorophyll fluorescence parameters), stable isotope (δ¹³C, δ¹⁵N) and metabolic carbon and nitrogen compounds. Generalists and specialists did not always differ in ecophysiological behaviour: for instance, while widespread generalists such as M. parvifolia and C. compressus were ecophysiologically versatile, R. brasiliense appeared to perform below optimum in all sampled habitats. Light and water were abiotic factors which apparently explained most of the variation found. Overall, ecophysiological responses were often more clearly related to individual species rather than to taxonomic affinities in higher hierarchies (family, genus) or habitat. These results are discussed as a further indication of high biodiversity even in habitats marginal to the rain forest hotspot.