María Josefa Rodríguez

Activation of the Fatty Acid α-Dioxygenase Pathway during Bacterial Infection of Tobacco Leaves FORMATION OF OXYLIPINS PROTECTING AGAINST CELL DEATH

A pathogen-induced oxygenase showing homology to prostaglandin endoperoxide synthases-1 and -2 was recently characterized by in vitro experiments as a fatty acid α-dioxygenase catalyzing formation of unstable 2(R)-hydroperoxy fatty acids. To study the activity of this enzyme under in vivo conditions and to elucidate the fate of enzymatically produced 2-hydroperoxides, leaves of tobacco were analyzed for the presence of α-dioxygenase-generated compounds as well as for lipoxygenase (LOX) products and free fatty acids. Low basal levels of 2-hydroxylinolenic acid (0.4 nmol/g leaves fresh weight) and 8,11,14-heptadecatrienoic acid (0.1 nmol/g) could be demonstrated. These levels increased strongly upon infection with the bacterium Pseudomonas syringae pv syringae (548 and 47 nmol/g, respectively). Transgenic tobacco plants overexpressing α-dioxygenase were developed, and incompatible infection of such plants led to a dramatic elevation of 2-hydroxylinolenic acid (1778 nmol/g) and 8,11,14-heptadecatrienoic acid (86 nmol/g), whereas the levels of LOX products were strongly decreased. Further analysis of oxylipins in infected leaves revealed the presence of a number of 2-hydroxy fatty acids differing with respect to chain length and degree of unsaturation as well as two new doubly oxygenated oxylipins identified as 2(R),9(S)-dihydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 2(R),9(S)-dihydroxy-10(E),12(Z)-octadecadienoic acid. α-Dioxygenase-generated 2-hydroxylinolenic acid, and to a lesser extent lipoxygenase-generated 9-hydroxyoctadecatrienoic acid, exerted a tissue-protective effect in bacterially infected tobacco leaves.

SADB phosphorylation of gamma-tubulin regulates centrosome duplication.

Symmetrical cell division requires duplication of DNA and protein content to generate two daughter cells. Centrosomes also duplicate during cell division, but the mechanism controlling this process is incompletely understood. We describe an alternative splice form of SadB encoding a short SADB Ser/Thr kinase whose activity fluctuates during the cell cycle, localizes to centrosomes, and controls centrosome duplication. Reduction of endogenous SADB levels diminished centrosome numbers, whereas enhanced SADB expression induced centrosome amplification. SADB exerted this action through phosphorylation of γ-tubulin on Ser 131, as expression of a phosphomimetic Ser 131-to-Asp γ-tubulin mutant alone increased centrosome numbers, whereas non-phosphorylatable Ala 131-γ-tubulin impaired centrosome duplication. We propose that SADB kinase activity controls centrosome homeostasis by regulating phosphorylation of γ-tubulin.

Cryo-X-ray tomography of vaccinia virus membranes and inner compartments

Vitrified unstained purified vaccinia virus particles have been used as a test sample to evaluate the capabilities of cryo-X-ray tomography. Embedded in a thick layer of vitreous ice, the viral particles representing the mature form of the virus (MV) were visualized using full-field transmission X-ray tomography. The tomographic reconstructions reveal the viral brick-shaped characteristic structures with a size of 250x270x360nm(3). The X-ray tomograms show the presence of a clearly defined external envelope, together with an inner core surrounded by an internal envelope, including areas with clear differential density, which correlate well with those features previously described for these viral particles using electron microscopy analyses. A quantitative assessment of the resolution attained in X-ray and electron tomograms of the viral particles prepared under the same conditions yields values of 25.7 and 6.7nm half-pitch, respectively. Although the resolution of the X-ray microscope is well above the dimensions of the membranous compartments, the strong differential contrast exhibited makes it possible to precisely reveal them without any contrasting reagent within this small and complex biological sample.

Cryo X-ray nano-tomography of vaccinia virus infected cells

Abstract We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.

Activation of a vinyl carbon–hydrogen bond in a tris(pyrazolyl)boratoiridium complex. The X-ray crystal structure of [IrH{HB(pz)3}(σ-C8H13)(η2-C8H14)]

Reaction between [(Ir(µ-Cl)(C8H14)2}2](C8H14= cyclo-octene) and Na[ HB(pz)](pz = pyrazolyl) affords the vinyl hydrido complex [IrH{HB(pz)3}(σ-C8H13)(η2-C8H14)]. Protonation of this complex gives [IrH{HB(pz)3}(η2-C8H14)2]BF4. Treatment of [IrCl(C2H4)4] with Na[HB(pz)3] yields [Ir{HB(pz)3)(η2-C2H4)2]. The molecular structure of [IrH{HB(pz)3}(σ-C8H13)(η2-C8H14)](1) has been determined by X-ray analysis. Crystals of (1) are monoclinic, space group P21/n, with a= 20.284(2), b= 12.265(1), c= 20.554(2)A, β= 97.103(7)°, and Z= 8. The co-ordination around the iridium atom is distorted octahedral involving the three N atoms from the tris(pyrazolyl) borato on one side, and a hydride, a π-bonded cycle-octene, and a π-bonded cyclo-octenyl group on the other; the last three ligands are mutually cis.

Membrane remodelling during vaccinia virus morphogenesis

Background information. VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components.

m-Xylene-Responsive Pu-PnifH Hybrid σ54 Promoters That Overcome Physiological Control in Pseudomonas putida KT2442

The sequences surrounding the -12/-24 motif of the m-xylene-responsive sigma54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH sigma54 promoter variants known to have various degrees of efficacy and affinity for sigma54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the -12/-24 region of the wild-type PnifH and its higher sigma54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.

Poly(1-pyrazolyl)boratoiridium and tris(1-pyrazolylmethyl)aminoiridium and -rhodium complexes

Abstract The synthesis of the Ir(H n BPz 4- n )(cod) ( n = 2, 0; Pz = pyrazolyl; cod = cycloocta -1,5-diene) complexes is described. They are prepared by reaction of [Ir(μ-Cl)(cod)] 2 with Tl(H n BPz 4- n ). The carbonyl compounds Ir(H n BPz 4- n )(CO) 2 ( n = 2, 0) are obtained by carbonylation of the cycloocta-1,5-diene derivatives. The preparations of the [Ir (coe) 2 {N(CH 2 Pz) 3 }]BF 4 (coe = cyclooctene) and [M(cod){N(CH 2 Pz) 3 }]BF 4 (M = Ir, Rh) compounds are also reported. They are prepared by treatment of [Ir(μ-Cl)(coe) 2 ] 2 or [M(μ-Cl)(cod)] 2 with N(CH 2 Pz) 3 and NaBF 4 . The carbonyl compound [Rh(CO) 2 {N (CH 2 Pz) 3 }]BF 4 is obtained by carbonylation of [Rh(cod){N(CH 2 Pz) 3 }]BF 4 .

Synthesis and nuclear magnetic resonance studies of some ethylene and ethyltris(pyrazolyl)boratoiridium complexes

Abstract The synthesis of [Ir{HB(pz)3}(C2H4)(CO)] (2), [Ir{HB(pz)3}(C2H5)(η2-C2H4)][BF4] (3), and [Ir{HB(pz)3}(C2H5)(CO)][BF4] (4) is described. Compound 2 is prepared by reaction of [Ir{HB(pz)3}(C2H4)2] (1) with carbon monoxide followed by treatment with ethylene under atmospheric pressure. Treatment of 1 and 2 with HBF4 affords compounds 3 and 4, respectively. Complex 2 is fluxional and its frozen structure is trigonal bipyramidal with the ethylene in the equatorial plane and the carbonyl group in one axial position; no rotation of the olefin is detected. Complesxes 3 and 4 are square-pyramidal and are rigid on the nuclear magnetic resonance (NMR) time scale. These structural inferences are based on 1H and 13C NMR spectra for the complexes and nuclear Overhauser effect (NOE) measurements for 2.

Carbon monoxide activation by poly(1-pyrazolyl)boratoiridium complexes

The syntheses of [IrH(HBPz3)(COOH)(CO)] (2), [IrH(HBPz3)(COOMe)(CO)] (5) and [IrH(HBPz3)-(COOEt)(CO)] (6) are described. They are prepared by the direct reaction of [Ir(HBPz3)(CO)2] (1) with H2O, MeOH, or ETOH, respectively. Compound (2) is stable in the solid state and in solution at room temperature, but decomposes in refluxing acetonitrile to give the dihydride [IrH2(HBPz3)(CO)] (3). The protonations of 1, 2 and 5 with HBF4·H2O lead to the cationic complex [IrH(HBPz3)(CO)2]BF4 (4). Complex 5 is also obtained by reaction of 2 with methanol, or by treatment of 4 with KOH/MeOH. The complex [Ir(BPz4)(CO)2] (7) also reacts with H2O and MeOH at room temperature, in the presence of CO; these reactions do not go to completion. The products of these reactions are most probably [IrH(BPz4)(COOH)(CO)] and [IrH(BPz4)(COOMe)(CO)]. Treatment of 7 with H2O in refluxing acetonitrile leads to [IrH2(BPz4)(CO)] (8).