Experimental infection of commercial layers with wild or attenuated Salmonella Gallinarum mutant strains: Anatomic pathology, total blood cell count and serum protein levels

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

Shear viscosity of a pion gas resulting from rho pi pi and sigma pi pi interactions

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

Rhamnolipids Production by a Pseudomonas eruginosa LBI Mutant: Solutions and Homologs Characterization

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

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Rho-omega mixing and the Nolen-Schiffer anomaly in relativistic nuclear models

We calculate within the framework of relativistic nuclear models the contribution of the ρ0 - ω mixing interaction to the binding energy differences of the mirror nuclei in the neighborhood of A = 16 and A = 40. We use two relativistic models for the nuclear structure, one with scalar and vector Woods-Saxon potentials, and the Walecka model. The ρ0 - ω interaction is treated in first order perturbation theory. When using the Walecka model the ρ- and ω-nucleon coupling constants are the same for calculating bound state wave functions and the perturbation due to the mixing. We find that the relativistic results on the average are of the same order as the ones obtained with nonrelativistic calculations.

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

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

Charge-symmetry breaking, rho-omega mixing, and the quark propagator

The momentum dependence of the ρ0-ω mixing contribution to charge-symmetry breaking (CSB) in the nucleon-nucleon interaction is compared in a variety of models. We focus in particular on the role that the structure of the quark propagator plays in the predicted behaviour of the ρ0-ω mixing amplitude. We present new results for a confining (entire) quark propagator and for typical propagators arising from explicit numerical solutions of quark Dyson-Schwinger equations We compare these to hadronic and free quark calculations The implications for our current understanding of CSB experiments is discussed.

rho(0) photoproduction in AuAu collisions at root s(NN)=62.4 GeV measured with the STAR detector

Vector mesons may be photoproduced in relativistic heavy-ion collisions when a virtual photon emitted by one nucleus scatters from the other nucleus, emerging as a vector meson. The STAR Collaboration has previously presented measurements of coherent rho(0) photoproduction at center of mass energies of 130 GeV and 200 GeV in AuAu collisions. Here, we present a measurement of the cross section at 62.4 GeV; we find that the cross section for coherent rho(0) photoproduction with nuclear breakup is 10.5 +/- 1.5 +/- 1.6mb at 62.4 GeV. The cross-section ratio between 200 GeV and 62.4 GeV is 4.4 +/- 0.6, less than is predicted by most theoretical models. It is, however, proportionally much larger than the previously observed 15% +/- 55% increase between 130 GeV and 200 GeV.

Characterization of the procera Tomato Mutant Shows Novel Functions of the SlDELLA Protein in the Control of Flower Morphology, Cell Division and Expansion, and the Auxin-Signaling Pathway during Fruit-Set and Development

procera (pro) is a tall tomato (Solanum lycopersicum) mutant carrying a point mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss of function, pro plants display a GA-constitutive response phenotype, mimicking wild-type plants treated with GA(3). The ovaries from both nonemasculated and emasculated pro flowers had very strong parthenocarpic capacity, associated with enhanced growth of preanthesis ovaries due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing self-pollination, similar to wild-type pistils treated with GA(3) or auxins. However, Style2.1, a gene responsible for long styles in noncultivated tomato, may not control the enhanced style elongation of pro pistils, because its expression was not higher in pro styles and did not increase upon GA(3) application. Interestingly, a high percentage of pro flowers had meristic alterations, with one additional petal, sepal, stamen, and carpel at each of the four whorls, respectively, thus unveiling a role of SlDELLA in flower organ development. Microarray analysis showed significant changes in the transcriptome of preanthesis pro ovaries compared with the wild type, indicating that the molecular mechanism underlying the parthenocarpic capacity of pro is complex and that it is mainly associated with changes in the expression of genes involved in GA and auxin pathways. Interestingly, it was found that GA activity modulates the expression of cell division and expansion genes and an auxin signaling gene (tomato AUXIN RESPONSE FACTOR7) during fruit-set.

Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42

Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to influence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knock-down strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not find a characteristic morphologic profile for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these findings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.

Immunohistochemical expression of Rho GTPases in ameloblastomas

Rho GTPases are proteins that regulate cell cycle, shape, polarization, invasion, migration, and apoptosis, which are important characteristics of normal and neoplastic cells. Rho GTPases expression has been reported in normal tooth germ and several pathologies; however, it has not been evaluated in ameloblastomas. The aim of this study was to analyze the expression and distribution of RhoA, RhoB, Rac1, and Cdc42 Rho GTPases in solid and unicystic ameloblastomas. Three-micrometer sections from paraffin- embedded specimens were evaluated by using an avidin- biotin immunohistochemical method with antibodies against the proteins mentioned above. RhoA and RhoB staining was observed in a high number of cells (P < 0.05) and greater intensity in non-polarized ones. Rac1 was not observed, andCdc42 didnot showany statistical differences between the number of non-polarized and basal positive cells (P > 0.05). Upon comparing the studied ameloblastomas, a higher number of positive cells in the unicystic variant was observed than that in the solid one (P < 0,05). The results obtained suggest that theseGTPases could play a role in the ameloblastoma neoplastic epithelial cell phenotype determination (polarized or non-polarized), as well as in variant (solid or unicystic) and subtype (follicular or plexiform) determination. Furthermore, they could participate in solid ameloblastoma invasion mechanisms. J Oral Pathol Med (2012) 41: 400-407

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

A dysflagellar mutant of Leishmania (Viannia) braziliensis isolated from a cutaneous leishmaniasis patient

Background: Parasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features. Methods: The parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo. Results: The isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis. Conclusions: Notwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FHR127H) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-gamma were able to synthesize FHR127H, the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-gamma secrete FH without retention in the ER. Moreover, the retention of FHR127H provoked enlargement of ER cisterns after treatment with IFN-gamma. A similar ER retention was observed in Cos-7 cells expressing the mutant FHR127H protein. Despite this deficiency in secretion, we show that the FHR127H mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein. The Journal of Immunology, 2012, 189: 3242-3248.

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.