Characterization of the atypical Ppz/Hal3 phosphatase system from the pathogenic fungus Cryptococcus neoformans

Ppz Ser/Thr protein phosphatases (PPases) are found only in fungi and have been proposed as potential antifungal targets. In Saccharomyces cerevisiae Ppz1 (ScPpz1) is involved in regulation of monovalent cation homeostasis. ScPpz1 is inhibited by two regulatory proteins, Hal3 and Vhs3, which have moonlighting properties, contributing to the formation of an unusual heterotrimeric PPC decarboxylase (PPCDC) complex crucial for CoA biosynthesis. Here we report the functional characterization of CnPpz1 (CNAG_03673) and two possible Hal3‐like proteins, CnHal3a (CNAG_00909) and CnHal3b (CNAG_07348) from the pathogenic fungus Cryptococcus neoformans. Deletion of CnPpz1 or CnHal3b led to phenotypes unrelated to those observed in the equivalent S. cerevisiae mutants, and the CnHal3b‐deficient strain was less virulent. CnPpz1 is a functional PPase and partially replaced endogenous ScPpz1. Both CnHal3a and CnHal3b interact with ScPpz1 and CnPpz1 in vitro but do not inhibit their phosphatase activity. Consistently, when expressed in S. cerevisiae, they poorly reproduced the Ppz1‐regulatory properties of ScHal3. In contrast, both proteins were functional monogenic PPCDCs. The CnHal3b isoform was crystallized and, for the first time, the 3D‐structure of a fungal PPCDC elucidated. Therefore, our work provides the foundations for understanding the regulation and functional role of the Ppz1‐Hal3 system in this important pathogenic fungus.     

Length polymorphisms at two candidate genes explain variation of migratory behaviors in blackpoll warblers (Setophaga striata)

Migratory behaviors such as the timing and duration of migration are genetically inherited and can be under strong natural selection, yet we still know very little about the specific genes or molecular pathways that control these behaviors. Studies in candidate genes Clock and Adcyap1 have revealed that both of these loci can be significantly correlated with migratory behaviors in birds, though observed relationships appear to vary across species. We investigated geographic genetic structure of Clock and Adcyap1 in four populations of blackpoll warblers (Setophaga striata), a Neotropical–Nearctic migrant that exhibits geographic variation in migratory timing and duration across its boreal breeding distribution. Further, we used data on migratory timing and duration, obtained from light‐level geolocator trackers to investigate candidate genotype–phenotype relationships at the individual level. While we found no geographic structure in either candidate gene, we did find evidence that candidate gene lengths are correlated with five of the six migratory traits. Maximum Clock allele length was significantly and negatively associated with spring arrival date. Minimum Adcyap1 allele length was significantly and negatively associated with spring departure date and positively associated with fall arrival date at the wintering grounds. Additionally, we found a significant interaction between Clock and Adcyap1 allele lengths on both spring and fall migratory duration. Adcyap1 heterozygotes also had significantly shorter migration duration in both spring and fall compared to homozygotes. Our results support the growing body of evidence that Clock and Adcyap1 allele lengths are correlated with migratory behaviors in birds.     

CpG/CpNpG motifs in the coding region are preferred sites for mutagenesis in the breast cancer susceptibility genes

The range of BRCA1/BRCA2 gene mutations is diverse and the mechanism accounting for this heterogeneity is obscure. To gain insight into the endogenous mutational mechanisms involved, we evaluated the association of specific sequences (i.e. CpG/CpNpG motifs, homonucleotides, short repeats) and mutations within the genes. We classified 1337 published mutations in BRCA1 (1765 BRCA2 mutations) for each specific sequence, and employed computer simulation combined with mathematical calculations to estimate the true underlying tendency of mutation occurrence. Interestingly, we found no mutational bias to homonucleotides and repeats in deletions/insertions and substitutions but striking bias to CpG/CpNpG in substitutions in both genes. This suggests that methylation-dependent DNA alterations would be a major mechanism for mutagenesis.     

Potential relationship between glutathione metabolism and flocculation in the yeast Kluyveromyces lactis

Reduced glutathione (GSH) is involved in biochemical and physiological processes in cells. Flocculation is an important mechanism in microorganisms. The present study concerned the potential relationship between GSH metabolism and flocculation. Two yeast strains, a flocculent (Kluyveromyces lactis 5c) and a nonflocculent (Kluyveromyces lactis 5a) strain, were used. The level of intracellular GSH measured during the growth period was significantly higher in the nonflocculent than in the flocculent strain; in contrast, the flocculent strain exhibited brighter staining of vacuoles than the nonflocculent strain when observed using epifluorescence microscopy. Compounds acting either on flocculation (EDTA, galactose) or on GSH metabolism (buthionine sulfoximine, and N-acetylcysteine) were tested on the flocculent strain during the growth period. Both EDTA and galactose fully inhibited flocculation and induced GSH overproduction of 58% and 153%, respectively. Buthionine sulfoximine decreased GSH level by 76% but had no effect on flocculation; N-acetylcysteine increased the GSH level and flocculation by 106% and 41%, respectively. Combination of EDTA and N-acetylcysteine produced similar effects than with each of them. Combination of galactose and N-acetylcysteine increased the GSH level but decreased flocculation. These results demonstrated that GSH homeostasis is linked to the flocculation mechanism. A hypothesis related to stress is given.     

A proteomics analysis of cell signaling alterations in colorectal cancer

To gain further insight into alterations in cellular pathways, tumor profiling, and marker discovery in colorectal cancer (CRC) we used a new antibody microarray specific for cell signaling. Soluble protein extracts were prepared from paired tumor/normal biopsies of 11 patients diagnosed with colorectal carcinoma at different stages; four liver carcinomas were used as a reference. Antibody microarray analysis identified 46 proteins that were differentially expressed between normal colorectal epithelium and adenocarcinoma. These proteins gave a specific signature for CRC, different from other tumors, as well as a panel of novel markers and potential targets for CRC. Twenty-four proteins were validated by using a specific colorectal cancer tissue microarray and immunoblotting analysis. Together with some previously well known deregulated proteins in CRC (beta-catenin, c-MYC, or p63), we found new potential markers preferentially expressed in CRC tumors: cytokeratin 13, calcineurin, CHK1, clathrin light chain, MAPK3, phospho-PTK2/focal adhesion kinase (Ser-910), and MDM2. CHK1 antibodies were particularly effective in discriminating between tumoral and normal mucosa in CRC. Moreover a global picture of alterations in signaling pathways in CRC was observed, including a significant up-regulation of different components of the epidermal growth factor receptor and Wnt/beta-catenin pathways and the down-regulation of p14(ARF). The experimental approach described here should be applicable to other pathologies and neoplastic processes.     

S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates

We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

     

Semisynthetic Triterpenes Derived from Euphorbia officinarum as Plant Growth Promoters and Inducers of Disease Resistance

This study evaluated the effect of application of the semisynthetic triterpenes 3β-acetoxy-norlup-20-one (F4) and 3-chloro-4α,14α-dimethyl-5α-cholest-8-ene (F6) triterpene derivatives from Euphorbia officinarum on the growth of tomato seedlings under normal conditions and when challenged with the pathogens Verticillium dahliae and Agrobacterium tumefaciens. Foliar spray of F4 and F6 significantly improved growth rate, fresh weight, dry weight, and leaf area. In addition, they enhanced several physiological parameters including photosynthetic pigments, proline content, and nitrate reductase activity. Moreover, they induced H₂O₂ accumulation and increased the activity of several antioxidant enzymes such as catalase, ascorbate peroxidase, and guaiacol peroxidase. They also enhanced disease resistance against V. dahliae and A. tumefaciens. These results suggest that the two semisynthetic triterpenes represent new plant growth regulators and inducers of plant disease resistance.

     

Endogenous VEGF-A is responsible for mitogenic effects of MCP-1 on vascular smooth muscle cells

Vessel wall remodeling is a complex phenomenon in which the loss of differentiation of vascular smooth muscle cells (VSMCs) occurs. We investigated the role of rat macrophage chemoattractant protein (MCP)-1 on rat VSMC proliferation and migration to identify the mechanism(s) involved in this kind of activity. Exposure to very low concentrations (1-100 pg/ml) of rat MCP-1 induced a significant proliferation of cultured rat VSMCs assessed as cell duplication by the counting of total cells after exposure to test substances. MCP-1 stimulated VSMC proliferation and migration in a two-dimensional lateral sheet migration of adherent cells in culture. Endogenous vascular endothelial growth factor-A (VEGF-A) was responsible for the mitogenic activity of MCP-1, because neutralizing anti-VEGF-A antibody inhibited cell proliferation in response to MCP-1. On the contrary, neutralizing anti-fibroblast growth factor-2 and anti-platelet-derived growth factor-bb antibodies did not affect VSMC proliferation induced by MCP-1. RT-PCR and Western blot analyses showed an increased expression of either mRNA or VEGF-A protein after MCP-1 activation (10-100 pg/ml), whereas no fms-like tyrosine kinase (Flt)-1 receptor upregulation was observed. Because we have previously demonstrated that hypoxia (3% O2) can enhance VSMC proliferation induced by VEGF-A through Flt-1 receptor upregulation, the effects of hypoxia on the response of VSMCs to MCP-1 were investigated. Severe hypoxia (3% O2) potentiated the growth-promoting effect of MCP-1, which was able to significantly induce cell proliferation even at a concentration as low as 0.1 pg/ml. These findings demonstrate that low concentrations of rat MCP-1 can directly promote rat VSMC proliferation and migration through the autocrine production of VEGF-A.     

Effect of Peroxides on [3H]d-Aspartate Release from Bovine Isolated Retinae

In the present study, we investigated the effect of naturally occurring and synthetic peroxides on K+-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae. Furthermore, effect of peroxides on endogenous glutamate concentrations were measured by HPLC in bovine neural retinae and vitreous humor of eyes treated with hydrogen peroxide (H2O2) ex vivo. Both naturally occurring H2O2 (1-100 microM) and synthetic (cumene hydroperoxide, cuOOH; 1-100 microM) peroxides caused a concentration-dependent inhibition of K+-evoked [3H]D-aspartate release without affecting basal tritium efflux. The antioxidant, trolox (2 mM) prevented the inhibition of evoked [3H]D-aspartate overflow elicited by both H2O2 (30 microM) and cuOOH (10 microM). Inhibition of catalase by 3-amino-triazole (3- AT 100 mM) enhanced an inhibitory effect of a low concentration of H2O2 (1 microM) but antagonized the effect of H2O2 (30 microM) on K+-induced [3H]D-aspartate release. In ex vivo experiments, exogenously applied H2O2 (1-100 microM) also caused a concentration-related decrease in glutamate levels in the bovine retina. We conclude that peroxides can inhibit K+-evoked release of [3H]D-aspartate and also decrease endogenous glutamate concentrations in the bovine retina.