Detection of the defoliating and nondefoliating pathotypes of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in artificial and natural soils by nested PCR

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.     

Phylogenetic relationships among some Ateles species: the use of chromosomic and molecular characters

As with most platyrrhines, the systematics of Ateles is under discussion. In order to help clarify its systematic, we employed chromosomic and molecular characters to analyze the phylogenetic relationship among some species of the genus Ateles. Chromosomic studies were conducted on 14 atelid specimens: eight Ateles from A. paniscus, A. chamek, A. belzebuth and A. geoffroyi, and six Alouatta caraya. Ateles paniscus showed 2N=32, whereas A. chamek, A. belzebuth and A. geoffroyi presented 2N=34, XX/XY (with a submetacentric X and a variable Y) corroborated by male meiosis. Nucleotide sequence variation at the mitochondrial cytochrome c oxidase subunit II gene (COII) was analyzed in ten New World monkey specimens. Parsimony trees showed consistent phylogenetic relationships using both chromosomic forms and mitochondrial COII gene sequences as characters. Particularly, chromosomic phylogenies showed A. hybridus as a divergent taxon from the remaining group, whereas A. chamek, A. belzebuth and A. marginatus form an unresolved clade with A. geoffroyi as sister group.     

Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions

The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169-180). Protein kinase C (PKC) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCgamma Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839-849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760-1765). This could be due to a failure of the mutant PKCgamma proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCgamma by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCgamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCgamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCgamma at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCgamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCgamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCgamma formed disulfide bonds in response to H2O2. H2O2-activated PKCgamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCgamma phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCgamma, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.     

The linker region joining the catalytic and the regulatory domains of CnA is essential for binding to NFAT

Calcineurin (CN) is an important regulator of developmental processes and in adults controls the immune response through its regulation of nuclear factor of activated T cells (NFAT). The physical interaction between CN and NFATs is an essential step in the activation of NFAT-dependent genes by calcium signals. Using deletional and substitutional analyses, we have identified a 13-amino acid region within CN that is essential for the interaction with NFAT and with two other CN-binding proteins, AKAP79 and Cabin-1. The interaction of CN with these proteins is selectively disrupted by substitution of specific amino acid residues within this region, indicating that NFAT and other CN-interacting proteins bind differentially to CN. This selectivity suggests that the region identified in CN could be a potential molecular target for immunosuppressive and other therapeutic interventions in diseases involving the CN/NFAT pathway.     

Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation

Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA-/- mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology.     

Downregulation of nNOS and synthesis of PGs associated with endotoxin-induced delay in gastric emptying

A single intraperitoneal injection of endotoxin (40 microg/kg) significantly delayed gastric emptying of a solid nutrient meal. Blockade of nitric oxide synthase (NOS) with 30 mg/kg ip N(G)-nitro-L-arginine methyl ester or 20 mg/kg ip 7-nitroindazole [neuronal NOS (nNOS) inhibitor] significantly delayed gastric emptying in control animals but failed to modify gastric emptying in endotoxin-treated rats. Administration of 2.5, 5, and 10 mg/kg ip N(6)-iminoethyl-L-lysine [inducible NOS (iNOS) inhibitor] had no effect in either experimental group. Indomethacin (5 mg/kg sc), NS-398 (cyclooxygenase-2 inhibitor; 10 mg/kg ip), and dexamethasone (10 mg/kg sc) but not quinacrine (20 mg/kg ip) significantly prevented delay in gastric emptying induced by endotoxin but failed to modify gastric emptying in vehicle-treated animals. Ca(2+)-dependent NOS activity in the antrum pylorus of the stomach was diminished by endotoxin, whereas Ca(2+)-independent NOS activity was not changed. In addition, decreased nNOS mRNA and protein were observed in the antrum pylorus of endotoxin-treated rats. Our results suggest that downregulation of nNOS in the antrum pylorus of the stomach and synthesis of prostaglandins mediate the delay in gastric emptying of a solid nutrient meal induced by endotoxin.     

Steps toward mapping the human vasculature by phage display

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.     

Gas chromatographic-mass spectrometry study of the essential oils of Pimenta racemosa var. terebinthina and P. racemosa var grisea

The essential oil of the leaves of P. racemosa var. terebinthina and P. racemosa var. grisea were examined by GC and GC/MS. The major constituents were alpha-terpineol acetate (27%), alpha-terpineol (20%) and 4-methoxy eugenol (12.6%) for Pracemosa var. terebinthina and 4-methoxy-isoeugenol (75.2%) and 4-methoxy-eugenol (4.5%) for P. racemosa var. grisea.