The effect of changes in the bulk dielectric constant on the DNA torsional properties

The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (K), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches K = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to K 1750 +/- 100. (c) 2007 Wiley Periodicals.

Eucalyptus ESTs corresponding to the enzyme glutamine synthetase and the protein D1, sites of action of herbicides that cause oxidative stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

Analysis of inteins in the Candida parapsilosis complex for simple and accurate species identification

Inteins are coding sequences that are transcribed and translated with flanking sequences and then are excised by an autocatalytic process. There are two types of inteins in fungi, mini-inteins and full-length inteins, both of which present a splicing domain containing well-conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes and may allow for the differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the three psilosis complex species C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work, we analyzed inteins that are present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of the Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis, and 22 C. orthopsilosis). Here, we describe an accessible and accurate technique based on a single PCR that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of the psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length forms in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies within C. orthopsilosis. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Glutathione and glutathione peroxidase expression in breast cancer: An immunohistochemical and molecular study

The use of prognostic markers for breast cancer allows therapeutic strategies to be defined more efficiently. The expression of glutathione (GSH) and glutathione peroxidase (GPX) in tumor cells has been evaluated as a predictor of prognosis and response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results showed that high GSH expression was associated with tumors negative for the estrogen receptor (ER) (P<0.05), and GPX expression was associated with tumors negative for the progesterone receptor (PR) and patient mortality. Focusing on the 37 patients who received adjuvant chemotherapy/radiotherapy (Group I), high expression of GPX was associated with a high rate of patient mortality (P<0.05). The 19 patients who received only adjuvant chemotherapy (Group II) showed high expression of GSH in relation to metastasis (P<0.05). In addition, high levels of GPX expression were significantly associated with a shorter overall survival (P<0.05). To confirm this, the expression of precursor genes of GSH [glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS)] and the GPX gene was analyzed using quantitative PCR in cultured neoplastic mammary cells treated with doxorubicin. Doxorubicin treatment was able to eliminate tumor cells without alterations in the gene expression of GSS, but led to underexpression of the GCLC and GPX genes. Our results suggest that high levels of GPX may be related to the development of resistance to chemotherapy in these tumors, response to treatment and the clinical course of the breast cancer patients.

Avaliação da glutationa e suas enzimas como marcadores prognósticos e preditivos do câncer de mama

Pós-graduação em Genética - IBILCE

Avaliação da glutationa e suas enzimas, e do estresse oxidativo como marcadores prognósticos e preditivos de neoplasia mamária em cadelas

Pós-graduação em Genética - IBILCE

Expression of glutathione, glutathione peroxidase and glutathione S-transferase pi in canine mammary tumors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoramento da expressão gênica de Xanthomonas axonopodis pv. citri em diferentes condições ambientais

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Physiological Studies on Candida albicans

Candida albicans is a common opportunistic, dimorphic human fungal pathogen. One of its virulence factors is the morphological switch between yeasts and hyphal or pseudohyphal forms, which can invade tissues and cause damage. Our studies focus on factors regulating pseudohyphae and epigenetic modifications of C. albicans. Regulating factors of pseudohyphae are aromatic alcohols and high phosphate. At low concentrations, exogenous aromatic alcohols induced pseudohyphae, as did high phosphate. For addressing the pathways involved in inducing pseudohyphae by aromatic alcohols or high phosphate, we used mutants defective in cAMP dependent PKA pathway (efg1/efg1), MAP kinase pathway (cph1/cph1), or both (cph1/cph1/efg1/efg1). These mutants failed to produce either hyphae or pseudohyphae in the presence of aromatic alcohols; but high phosphate still stimulated pseudohyphae. Gcn4, a transcription activator of more than 500 amino acid related genes, is turned-on in response to amino acid starvation. The accumulation of aromatic alcohols sends nitrogen starvation signals, which inhibit eIF2B, which in turn derepresses Gcn4p. High phosphate also induces pseudohyphae by derepressing Gcn4p, although the pathways involved are still unknown. In sum, aromatic alcohols and high phosphate induce pseudohyphae by derepressing Gcn4. In this study we found a novel posttranslational histone modification in C. albicans, which is biotinylation. Western blot and Mass spectrometry techniques were used to find that Histones H2B and H4 were biotinylated at every condition tested such as yeast vs. hyphae, aerobic growth vs. anaerobic growth, rich medium vs. defined medium. In C. albicans lysines K8, K11 in histone H4 and lysines K17, K18, K31 in histone H2B are biotin attachment sites as found using mass spectrometry. Biotin was also found to enhance the germ tube formation of C. albicans. Germ tube formation assays with biotin-starved cells as inoculum showed low percent of germ tubes (1-5%). Addition of biotin to the media showed 100% germ tubes. Biotinylation of histones were not detected from biotin-starved cells. Appendix-A details work related to Farnesol quantification assays in several strains of C.albicans and Ceratocystis ulmi, and growth studies of class E VPS strains of Saccharomyces Cerevisiae. Adviser: Kenneth W. Nickerson

Holocarboxylase Synthetase-dependent Biotinylation of Histone H4

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysine (K) residues in histones H3 and H4. Histone biotinylation marks play important roles in the repression of genes and retrotransposons. Preliminary studies suggested that K16 in histone H4 is a target for biotinylation by HCS. Here we demonstrated that H4K16bio is overrepresented in repeat regions {pericentromeric alpha satellite repeats; long terminal repeats (LTR)} compared with euchromatin promoters. H4K16bio was also enriched in the repressed interleukin-2 gene promoter. The enrichment at LTR22 and promoter 1 of the sodium-dependent multivitamin transporter (SMVT) depended on biotin supply; and was significantly lower in fibroblasts from an HCS-deficient patient compared with an HCS wild-type control. We conclude that H4K16bio is a real phenomenon and plays a role in the transcriptional repression of repeats and genes. HCS catalyzes the covalent binding of biotin to carboxylases, in addition to its role as a histone biotinyl ligase. HCS null individuals are not viable whereas HCS deficiency is linked to developmental delays and phenotypes such as short life span and low stress resistance. Here, we developed a 96-well plate assay for high-throughput analysis of HCS based on the detection of biotinylated p67 using IRDye-streptavidin and infrared spectroscopy. We demonstrated that the catalytic activity of rHCS depends on temperature and time, and proposed optimal substrate and enzyme concentrations to ensure ideal measurement of rHCS activity and its kinetics. Additionally, we demonstrated that this assay is sensitive enough to detect biotinylation of p67 by endogenous HCS from Jurkat lymphoid cells.

Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt

Goncalves DA, Silveira WA, Lira EC, Gra a FA, Paula-Gomes S, Zanon NM, Kettelhut IC, Navegantes LC. Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt. Am J Physiol Endocrinol Metab 302: E123-E133, 2012. First published September 27, 2011; doi:10.1152/ajpendo.00188.2011.-Although it is well known that administration of the selective beta(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 mu M), a PKA activator. The in vitro addition of triciribine (10 mu M), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous cofactor participating in numerous redox reactions. It is also a substrate for regulatory modifications of proteins and nucleic acids via the addition of ADP-ribose moieties or removal of acyl groups by transfer to ADP-ribose. In this study, we use in-depth sequence, structure and genomic context analysis to uncover new enzymes and substrate-binding proteins in NAD-utilizing metabolic and macromolecular modification systems. We predict that Escherichia coli YbiA and related families of domains from diverse bacteria, eukaryotes, large DNA viruses and single strand RNA viruses are previously unrecognized components of NAD-utilizing pathways that probably operate on ADP-ribose derivatives. Using contextual analysis we show that some of these proteins potentially act in RNA repair, where NAD is used to remove 2'-3' cyclic phosphodiester linkages. Likewise, we predict that another family of YbiA-related enzymes is likely to comprise a novel NAD-dependent ADP-ribosylation system for proteins, in conjunction with a previously unrecognized ADP-ribosyltransferase. A similar ADP-ribosyltransferase is also coupled with MACRO or ADP-ribosylglycohydrolase domain proteins in other related systems, suggesting that all these novel systems are likely to comprise pairs of ADP-ribosylation and ribosylglycohydrolase enzymes analogous to the DraG-DraT system, and a novel group of bacterial polymorphic toxins. We present evidence that some of these coupled ADP-ribosyltransferases/ribosylglycohydrolases are likely to regulate certain restriction modification enzymes in bacteria. The ADP-ribosyltransferases found in these, the bacterial polymorphic toxin and host-directed toxin systems of bacteria such as Waddlia also throw light on the evolution of this fold and the origin of eukaryotic polyADP-ribosyltransferases and NEURL4-like ARTs, which might be involved in centrosomal assembly. We also infer a novel biosynthetic pathway that might be involved in the synthesis of a nicotinate-derived compound in conjunction with an asparagine synthetase and AMPylating peptide ligase. We use the data derived from this analysis to understand the origin and early evolutionary trajectories of key NAD-utilizing enzymes and present targets for future biochemical investigations.