Transcriptional and Metabolic Changes Associated to the Infection by Fusarium verticillioides in Maize Inbreds with Contrasting Ear Rot Resistance

Fusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.     

Short acidic peptides isolated from wheat sprout chromatin and involved in the control of cell proliferation. Characterization by infrared spectroscopy and mass spectrometry

Low molecular weight peptides were isolated from the chromatin of wheat sprouts. Following gel filtration the peptide fraction shows a sharp inhibiting activity on the growth of HeLa cancer cells. Infrared (IR) spectroscopy and mass spectrometry have been utilized to characterize the wheat sprout peptides in an attempt to recognize the peptide sequence involved in the control of cell growth. The quantitative presence of a peptide with MH+=572 appears proportional to the cell growth inhibition activity. This compound has been subjected to extensive mass spectrometry analysis. The automatic computational analysis of the ions of second, third and fourth generations indicate a peptide sequence, AcHis-Asp-Ser-Glu-, that binds at the C-terminal a molecule of ethanolamine. Moreover, the results show that some sequences of the wheat sprout peptide family are present in the peptide fractions isolated from several other tissues, thus supporting the hypothesis of ubiquitous regulatory peptides.     

Vanadium (V) peroxocomplexes: structure, chemistry and biological implications

A short account on the identification in solution of diverse type of vanadium peroxocomplexes is offered. The methodology used is a combination of several techniques, i.e., multinuclear NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and theoretical calculation. The analysis has been carried out in aqueous alcoholic solutions, and in some case also in the presence of appropriate ligands, in order to model some of the natural conditions where vanadium-dependent haloperoxidase enzymes (VHPO) work. With the results obtained, it has been possible to shed light on important aspects of the catalytic cycle of VHPO. Furthermore, a number of synthetic aspects of the reactivity of the various vanadium peroxocomplexes is reviewed.     

The oxytocin receptor antagonist atosiban inhibits cell growth via a "biased agonist" mechanism

In human myometrial cells, the promiscuous coupling of the oxytocin receptors (OTRs) to G(q) and G(i) leads to contraction. However, the activation of OTRs coupled to different G protein pathways can also trigger opposite cellular responses, e.g. OTR coupling to G(i) inhibits, whereas its coupling to G(q) stimulates, cell proliferation. Drug analogues capable of promoting a selective receptor-G protein coupling may be of great pharmacological and clinical importance because they may target only one specific signal transduction pathway. Here, we report that atosiban, an oxytocin derivative that acts as a competitive antagonist on OTR/G(q) coupling, displays agonistic properties on OTR/G(i) coupling, as shown by specific (35)S-labeled guanosine 5'-3-O-(thio) trisphosphate ([(35)S]GTPgammaS) binding. Moreover, atosiban, by acting on a G(i)-mediated pathway(,) inhibits cell growth of HEK293 and Madin-Darby canine kidney cells stably transfected with OTRs and of DU145 prostate cancer cells expressing endogenous OTRs. Notably, atosiban leads to persistent ERK1/2 activation and p21(WAF1/CIP1) induction, the same signaling events leading to oxytocin-mediated cell growth inhibition via a G(i) pathway. Finally, atosiban exposure did not cause OTR internalization and led to only a modest decrease (20%) in the number of high affinity cell membrane OTRs, two observations consistent with the finding that atosiban did not lead to any desensitization of the oxytocin-induced activation of the G(q)-phospholipase C pathway. Taken together, these observations indicate that atosiban acts as a "biased agonist" of the human OTRs and thus belongs to the class of compounds capable of selectively discriminating only one among the multiple possible active conformations of a single G protein-coupled receptor, thereby leading to the selective activation of a unique intracellular signal cascade.     

Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope

Increasing evidence points to a dynamical compartmentalization of the cell nucleus, yet the mechanisms by which interphase chromatin moves and is positioned within nuclei remain unclear. Here, we study the dynamics of chromatin in vivo applying a novel particle-tracking method in a two-photon microscope that provides approximately 10-fold higher spatial and temporal resolutions than previous measurements. We followed the motion of a chromatin sequence containing a lac-operator repeat in cells stably expressing lac repressor fused with enhanced green fluorescent protein, observing long periods of apparent constrained diffusion interrupted by relatively abrupt jumps of approximately 150 nm lasting 0.3-2 s. During these jumps, the particle moved an average of four times faster than in the periods between jumps and in paths more rectilinear than predicted for random diffusion motion. Additionally, the jumps were sensitive to the temperature and absent after ATP depletion. These experimental results point to an energy-dependent mechanism driving fast motion of chromatin in interphase cells.     

A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines

The cAMP protein kinase A (PKA) pathway in T cells conveys an inhibitory signal to suppress inflammation. This study was performed to understand the mechanisms involved in cAMP-mediated signaling in T lymphocytes. A-kinase anchoring proteins (AKAPs) bind and target PKA to various subcellular locations. AKAPs also bind other signaling molecules such as cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cAMP in the cell. PDE4 and PDE7 have important roles in T cell activation. Based on this information, we hypothesized that AKAPs associate with PDEs in T lymphocytes. Immunoprecipitation of Jurkat cell lysates with Abs against both the regulatory subunit of PKA (RIIalpha) and specific AKAPs resulted in increased PDE activity associated with RIIalpha and AKAP95, AKAP149, and myeloid translocation gene (MTG) compared with control (IgG). Immunoprecipitation and pull-down analyses demonstrate that PDE4A binds to AKAP149, AKAP95, and MTG, but not AKAP79, whereas PDE7A was found to bind only MTG. Further analysis of MTG/PDE association illustrated that PDE4A and PDE7A bind residues 1-344 of MTG16b. Confocal analysis of HuT 78 cells stained with anti-PDE7A showed overlapping staining patterns with the Golgi marker GM130, suggesting that PDE7A is located in the Golgi. The staining pattern of PDE7A also showed similarity to the staining pattern of MTG, supporting the immunoprecipitation data and suggesting that MTG may interact with PDE7A in the Golgi. In summary, these data suggest that AKAPs interact with both PKA and PDE in T lymphocytes and thus are a key component of the signaling complex regulating T cell activation.     

DNA fragmentation, DNA repair and apoptosis induced in primary rat hepatocytes by dienogest, dydrogesterone and 1,4,6-androstatriene-17beta-ol-3-one acetate

Four steroids that share the 17-hydroxy-3-oxopregna-4,6-diene structure - cyproterone acetate, chlormadinone acetate, megestrol acetate, and potassium canrenoate - have been shown previously to behave with different potency as liver-specific genotoxic agents, the response being markedly higher in female than in male rats, but similar in humans of both genders. In this study, performed to better define the relationship between chemical structure and genotoxicity, dydrogesterone (DGT) with double bonds C4=C5 and C6=C7, dienogest (DNG) with double bonds C4=C5 and C9=C10, and 1,4,6-androstatriene-17beta-ol-3-one acetate (ADT) with double bonds C1=C2, C4=C5 and C6=C7, were compared with cyproterone acetate (CPA) for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from three rats of each sex. At subtoxic concentrations, ranging from 10 to 90 microM, all four steroids consistently induced a dose-dependent increase of DNA fragmentation, which in all cases was higher in females than in males; their DNA damaging potency decreased in the order CPA > DNG > ADT > DGT. Under the same experimental conditions, the responses provided by the DNA repair-synthesis assay were positive or inconclusive in hepatocytes from female rats and consistently negative in hepatocytes from male rats. In the induction of apoptotic cells, examined in primary hepatocytes from female rats, CPA was more active than ADT and DGT, and DNG was inactive. Considered as a whole these findings suggest that a liver-specific genotoxic effect more marked in female than in male rats might be a common property of steroids with two or three double bonds.