In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.     

Physiology of lipase formation inPenicillium candidum

Penicillium candidum grew and produced lipase in a culture medium supplemented with 0.2% olive oil. Significant enzyme production required the presence of olive, oil and was prevented by cycloheximide. Polyacrylamide gel electrophoresis of filtrates from olive oil fermentations gave a single band of lipase activity (MW 80 KDa). Among the olive oil components only oleate allowed significant lipase production. Other carboxylic and saturated fatty acids containing similar or lower numbers of carbon atoms, did not cause derepression of lipase formation.     

Coding of odor intensity in a steady-state deterministic model of an olfactory receptor neuron

The coding of odor intensity by an olfactory receptor neuron model was studied under steady-state stimulation. Our model neuron is an elongated cylinder consisting of the following three components: a sensory dendritic region bearing odorant receptors, a passive region consisting of proximal dendrite and cell body, and an axon. First, analytical solutions are given for the three main physiological responses: (1) odorant-dependent conductance change at the sensory dendrite based on the Michaelis-Menten model, (2) generation and spreading of the receptor potential based on a new solution of the cable equation, and (3) firing frequency based on a Lapicque model. Second, the magnitudes of these responses are analyzed as a function of odorant concentration. Their dependence on chemical, electrical, and geometrical parameters is examined. The only evident gain in magnitude results from the activation-to-conductance conversion. An optimal encoder neuron is presented that suggests that increasing the length of the sensory dendrite beyond about 0.3 space constant does not increase the magnitude of the receptor potential. Third, the sensivities of the responses are examined as functions of (1) the concentration at half-maximum response, (2) the lower and upper concentrations actually discriminated, and (3) the width of the dynamic range. The overall gain in sensitivity results entirely from the conductance-to-voltage conversion. The maximum conductance at the sensory dendrite appears to be the main tuning constant of the neuron because it determines the shift toward low concentrations and the increase in dynamic range. The dynamic range of the model cannot exceed 5.7 log units, for a sensitivity increase at low odor concentration is compensated by a sensitivity decrease at high odor concentration.     

Relationships between carbon and hydrogen isotope ratios and nitrogen levels in leaves ofClusia species and two other Clusiaceae genera at various sites and different altitudes in Venezuela

Samples of the Clusiaceae generaClusia, Oedematopus andDystovomita were collected at various sites and different altitudes in northern and south-western Venezuela. Analyses of stable isotopes of carbon and hydrogen and of leaf-nitrogen levels were performed on the dried samples. Correlations among these variables, i.e. carbon isotope discrimination (), hydrogen isotope ratio (D) and N-levels, and with altitude were assessed. In the samples, where values of above 15 indicate predominant performance of C₃ photosynthesis, there were slight tendencies of increasing , D and N-levels with increasing altitude and of increasing with increasing N. Although these correlations taken separately were not statistically significant, they support each other and indicate increasing transpiration and increased leaf-nutrient supply at increasing altitude. Performance of crassulacean acid metabolism (CAM) in species ofClusia appears to be restricted to altitudes below 1500 m a.s.l. There was a significant negative correlation of with altitude in the samples, where values of below 10 indicated predominant performance of CAM. This suggests that phases II and IV of CAM are progressively suppressed towards the upper altitudinal limit of CAM inClusia in northern Venezuela. It is concluded that among the large number of environmental factors and combinations thereof, which determine the expression of CAM inClusia and trigger C₃-CAM transitions in C₃/CAM intermediate species, low availability of water is the most important.     

Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae

An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide-binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild-type strain and we investigated the response of the various mutants to treatment with synthetic competence-stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide-binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide-binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two-component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid-insertion mutation abolished competence completely. In addition, the triple AmiA-AliA-AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.     

Spatial structure of mitochondria and ER denotes changes in cell physiology of cultured tobacco protoplasts

The structure of mitochondria and of the endoplasmic reticulum (ER) in mesophyll protoplasts and regenerated cells was studied in vivo using the dye DiOC₆(3) (3,3'-dihexyloxacarbocyanine iodide) and confocal laser scanning microscopy (CLSM). The relation to the cell's physiology was investigated using a hormone-based model system for elongation and division. The structure of the mitochondria and of their population depends on the status of the cell. In freshly isolated protoplasts small spherical mitochondria are clustered around the nucleus and the chloroplasts. During the first 4 days of culture they are transformed into long vermiform organelles which distribute evenly throughout the cytoplasm. In a medium containing only auxins, cells then enter a period of expansion. Their mitochondria retain the same structure but increase in quantity. In a medium with auxins and cytokinins cells start dividing. Their mitochondria typically become numerous and very small, and are distributed throughout the cytoplasm. Both types of organization were observed during weeks of ongoing expansion or division. The ER is always present as a network close to the cell membrane. In freshly isolated protoplasts a considerable part of the ER is clustered around the chloroplasts, the remaining part of the network has a reduced complexity and is partly broken. During subsequent protoplast culture the network grows into a complex web with fine meshes incorporating lots of plate-like structures. This is the case in elongating cells as well as in dividing cells. Finally, the ER looks similar to the structure found in epidermal cells of the intact plant.     

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

Introduction of the pea (Pisum sativum L.) lectin (PSL) gene into white clover (Trifolium repens L.) hairy roots facilitates nodulation by the nitrogen-fixing bacterium Rhizobium leguminosarum biovar viciae, which normally nodulates pea and not white clover (C.L. Diaz, L.S. Melchers, P.J.J. Hooykaas, B.J.J. Lugtenberg, and J.W. Kijne [1989] Nature 338: 579-581). Here, we show that PSL is functionally expressed in transgenic white clover hairy roots transformed with the PSL gene. PSL could be isolated from these roots by affinity chromatography. Immunoanalysis of PSL showed the presence of polypeptides corresponding to the PSL precursor and its [beta] subunits. In addition, we developed a highly sensitive localization technique based on specific binding of a glycan moiety of rat IgE to PSL. Similar to the situation in pea roots, PSL appeared to be localized on the external cell surface of elongated epidermal cells and on the tips of emerging and growing root hairs of transgenic white clover hairy roots. PSL was not observed on normal white clover roots and on hairy roots without the PSL gene. These results show that (a) in transgenic white clover hairy roots, PSL is correctly processed and targeted to root cells susceptible to rhizobial infection, and (b) like in pea roots, PSL is surface bound with at least one of its two sugar-binding sites available for (rhizobial) ligands.     

Structural modifications of oxidized insulin B-chain induced by glycerol addition

H-NMR studies of the bovine insulin S-sulfonatedB-chain are reported in H₂O/D₂O (9/1) and inglycerol-d₅ (5 M) using two-dimensional NMRspectroscopy. The first results show that the oxidizedinsulin B-chain secondary structure differs from thatof native insulin by a loss of the -helixbetween the two disulfide bridges and that theglycerol favours the structuring of the peptide.     

Influence of Virus on the Chlorophyll, Carotenoid and Polyamine Contents in Grapevine Microcuttings

We have studied the effect of different types of virus infections on the content in chlorophylls, carotenoids and free polyamines in shoots of vine cv. Albariño cultured in vitro. The ratio of chlorophyll a/c hlorophyll b was maintained practically constant in the different cases studied. A significant increase was observed in the polyamines, especially putrescine, in shoots infected with the different types of virus, mainly in the cases of grapevine fleck disease+grapevine leafroll disease type I and grapevine stem pitting.