Apoptosis and cell recovery in response to oxidative stress in p53-deficient prostate carcinoma cells

We have studied the effects of different concentrations of H(2)O(2) on the proliferation of PC-3 prostate carcinoma cells. Since this cell line lacks functional p53, we sought to characterize whether apoptotic response to the oxidative insult was altered such that, unlike in cells containing functional p53 apoptosis may be reduced and replaced by other mechanisms of cellular arrest and death. We did not observe necrosis in PC-3 cells treated with H(2)O(2) concentrations of up to 500 microM. In the presence of 50 microM H(2)O(2), arrest was observed in the G2-phase of the cell cycle, along with p53-independent apoptosis. In the presence of 500 microM H(2)O(2), addition of l-buthionine sulfoximine increased the percentage of apoptotic cell death. Senescence-associated cell arrest was never observed. Moreover, some of the treated cells seemed to be resistant to oxidative damage. These cells re-entered the cell cycle and proliferated normally. Analysis of the expression of p21(waf1) and of p21 protein levels, as well as the activity of caspase-3 and caspase-8, allowed us to characterize some aspects of the arrest of PC-3 cells in G2 and the apoptotic response to oxidative stress in the absence of functional p53.     

Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola

A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola.     

Conservation of key members in the course of the evolution of the insulin signaling pathway

Our understanding of the evolution of the insulin signaling pathway (ISP) is still incomplete. One intriguing unanswered question is the explanation of the emergence of the glucostatic role of insulin in mammals. To find out whether this is due to the development of new sets of signaling transduction elements in these organisms, or to the establishment of new interactions between pre-existing proteins, we rebuilt putative orthologous ISPs in 17 eukaryotic organisms. Then, we computed the conservation of orthologous ISPs at different levels, from sequence similarity of orthologous proteins to co-evolution of interacting domains. We found that the emergence of glucostatic role in mammals can neither be explained by the development of new sets of signaling elements, nor by the establishment of new interactions between pre-existing proteins. The comparison of orthologous IRS molecules indicates that only in mammals have they acquired their complete functionality as efficient recruiters of effector sub-pathways.     

beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophic fungus Botrytis cinerea

The non-protein amino acid beta-aminobutyric acid (BABA) protects numerous plants against various pathogens. Protection of Arabidopsis plants against virulent pathogens involves the potentiation of pathogen-specific defense responses. To extend the analysis of the mode of action of BABA to necrotrophs we evaluated the effect of this chemical on Arabidopsis plants infected with the gray mold fungus Botrytis cinerea. BABA-treated Arabidopsis were found to be less sensitive to two different strains of this pathogen. BABA protected mutants defective in the jasmonate and ethylene pathways, but was inactive in plants impaired in the systemic acquired resistance transduction pathway. Treatments with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester, a functional analog of salicylic acid (SA), also markedly reduced the level of infection. Moreover, BABA potentiated mRNA accumulation of the SA-associated PR-1, but not the jasmonate/ethylene-dependent PDF1.2 gene. Thus, besides jasmonate/ethylene-dependent defense responses, SA-dependent signaling also contributes to restrict B. cinerea infection in Arabidopsis. Our results also suggest that SA-dependent signaling is down-regulated after infection by B. cinerea. The observed up-regulation of the PDF1.2 gene in mutants defective in the SA-dependent signaling pathway points to a cross-talk between SA- and jasmonate/ethylene-dependent signaling pathways during pathogen ingress.     

Chloroplast nitrite uptake is enhanced in Arabidopsis PII mutants

In higher plants, the PII protein is a nuclear-encoded plastid protein that regulates the activity of a key enzyme of arginine biosynthesis. We have previously observed that Arabidopsis PII mutants are more sensitive to nitrite toxicity. Using intact chloroplasts isolated from Arabidopsis leaves and (15)N-labelled nitrite we show that a light-dependent nitrite uptake into chloroplasts is increased in PII knock-out mutants when compared to the wild-type. This leads to a higher incorporation of (15)N into ammonium and amino acids in the mutant chloroplasts. However, the uptake differences do not depend on GS/GOGAT activities. Our observations suggest that PII is involved in the regulation of nitrite uptake into higher plant chloroplasts.     

Genetic diversity in Spanish donkey breeds using microsatellite DNA markers

Genetic diversity at 13 equine microsatellite loci was compared in five endangered Spanish donkey breeds: Andaluza, Catalana, Mallorquina, Encartaciones and Zamorano-Leonesa. All of the equine microsatellites used in this study were amplified and were polymorphic in the domestic donkey breeds with the exception of HMS1, which was monomorphic, and ASB2, which failed to amplify. Allele number, frequency distributions and mean heterozygosities were very similar among the Spanish donkey breeds. The unbiased expected heterozygosity (H_E) over all the populations varied between 0.637 and 0.684 in this study. The low G_ST value showed that only 3.6% of the diversity was between breeds (P < 0.01). Significant deviations from Hardy-Weinberg equilibrium were shown for a number of locus-population combinations, except HMS5 that showed agreement in all analysed populations. The cumulative exclusion probability (PE) was 0.999 in each breed, suggesting that the loci would be suitable for donkey parentage testing. The constructed dendrogram from the D_A distance matrix showed little differentiation between Spanish breeds, but great differentiation between them and the Moroccan ass and also with the horse, used as an outgroup. These results confirm the potential use of equine microsatellite loci as a tool for genetic studies in domestic donkey populations, which could also be useful for conservation plans.     

Nucleolar Organizer ultrastructure in Allium cepa

The Nuoleolar Organizer Region (NOR) was studied in Allium cepa. The cells analyzed were: meristematic root tip cells, cells from the layers of the anther (exothecium, intermediate layer cells, endothecium and tapetum) and meiocytes from preleptotene to diptotene. Conventional electron microscope techniques and serial section were used in the study. Ultracytochemical tests, using preferential techniques for nucleic acids were also applied. The results show that: a) Although the NOR may have a very variable form in the different cells analyzed, in each case its fine structure is similar, being constituted of chromatin differentiated in high and low density zones. b) In each case, its cytochemical characteristics are similar; highly positively to EDTA (preferential stain for RNP) being observed in the low density zones. As scattered chromatin these would constitute the active regions of the NOR. c) In certain cases a clear relationship is seen between NOR volume and the size of the nucleolus. d) The chromatin associated to the NOR always appears as very dense zones. e) The NOR-Nucleolus relationships are established exclusively with the latter's fibrillar pars. f) The nucleolar segregation phenomenon takes place sometimes as a result of a NOR contraction and in other cases depends on the position of the NOR in relation to the nucleolus. g) In preleptotene, the NOR shows its characteristic morphology and is positive to EDTA, while from zygotene to diplotene the structure of the NOR presents no differences from the rest of the chromatin and only the lateral elements of the synaptonemal complex or its remnants are positive to EDTA. h) The synaptonemal complex runs through the NOR with no differentiation and on occasions its lateral elements show associations with the nucleolus.