Genes diferencialmente expressos em cana-de-açúcar inoculada com Xanthomonas albilineans, o agente causal da escaldadura da folha

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

Association between MUC1 gene polymorphism and expected progeny differences in Nelore cattle (Bos primigenius indicus)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effects of polymorphic microsatellites in the regulatory region of IGF1 and GHR on growth and carcass traits in beef cattle

Growth hormone (GH), insulin-like growth factors 1 and 2 (IGF1 and IGF2) and their associated binding proteins and transmembrane receptors (GHR, IGF1R and IGF2R) play an important role in the physiology of mammalian growth. The objectives of the present study were to estimate the allele and genotype frequencies of microsatellite markers located in the 5'-regulatory region of the IGF1 and GHR genes in beef cattle belonging to different genetic groups and to determine effects of these markers on growth and carcass traits in these animals under an intensive production system. For this purpose, genotyping was performed on 384 bulls including 79 Nellore, 30 Canchim (5/8 Charolais + 3/8 Zebu) and 275 crossbred animals originating from crosses of Simmental (1/2 Simmental, n = 30) and Angus (1/2 Angus, n = 245) sires with Nellore females. The effects of substituting L allele for S allele of GHR microsatellite across Nellore, Canchim and 1/2 Angus were significant for weight gain and body weight (P < 0.05). The IGF1 microsatellite allele substitutions of 229 for 225 within Nellore group and of 225 for 229 within 1/2 Angus were not significant for any of the traits.

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P(4)). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25 degrees C, were incubated for 30 min at 38 degrees C in 5% CO(2) and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P(4) was added (10 mu g/ml) to the P1 group. Groups P2 and P3 were supplemented with P(4) (10 and 20 mu g/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 mu M, respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P(4) treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P(4) groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 mu M seems to be more suitable to trigger AR in domestic cat sperm.

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

Aquaporin 9 (AQP9) localization in the adult dog testis excurrent ducts by immunohistochemistry

Aquaporins (AQPs) are small, intrinsic membrane proteins that are present in many cell types involved in fluid transport. AQP9 is a major apical water channel that is expressed throughout the efferent ducts, epididymis, and vas deferens, as well as in other regions of the human and rodent male reproductive tract. The target of this study was to examine the expression of AQP9 in epithelial cells in the adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis; initial segment, caput, corpus, and cauda of the epididymis; and vas deferens were obtained from eight adult mongrel dogs. Immunohistochemistry and Western blotting procedures were used to show AQP9 localization and distribution. AQP9 expression was not detected either in dog seminiferous tubules or rete testis. However, apical labeling for AQP9 was detected in the different regions of epididymis and vas deferens, with the reaction being less intense in the caput epididymis. Thus, AQP9 is abundantly expressed in dog male reproductive tract, in which it is an important apical pathway for transmembrane flow of water and neutral solutes.

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133(+) stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10 (13) mol iron (9.5 pg) or 7.0 x 10 (6) nanoparticles per cell ( the measurement was carried out in a volume of 2 mu L containing about 6.16 x 10 5 pg iron, equivalent to 4.5 x 10 (11) SPIONs). (c) 2008 Elsevier B.V. All rights reserved.

In vitro and in vivo studies on CCR10 regulation by Annexin A1

The mode of action of annexin A1 (ANXA1) is poorly understood. By using rapid subtraction hybridization we studied the effects of human recombinant ANXA1 and the N-terminal ANXA1 peptide on gene expression in a human larynx cell line. Three genes showed strong downregulation after treatment with ANXA1. In contrast, expression of CCR10, a seven transmembrane G-protein coupled receptor for chemokine CCL27 involved in mucosal immunity, was increased. Moreover the reduction in CCR10 expression induced by ANXA1 gene deletion was rescued by intravenous treatment with low doses of ANXA1. These findings provide new evidence that ANXA1 modulates gene expression. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Demulsification of water/sunflower oil emulsions by a tangential filtration process using chemically impregnated ceramic tubes

A tangential filtration process was implemented in this study using porous ceramic tubes made of alpha-alumina produced by the slip-casting technique. These tubes were sintered at 1450 degrees C and characterized by mercury intrusion porosimetry, which revealed a mean pore size of 0.5 mu m. The tubes were chemically impregnated with a zirconium citrate solution, after which they were calcined and heat treated at temperatures of up to 600 and 900 degrees C to eliminate volatile organic compounds and transform the zirconium citrate into zirconium oxide impregnated in the alumina in the form of nanoparticle agglomerates. The microporous pipes were tested on a microfiltration hydraulic system to analyze their performance in the demulsification of sunflower oil and water mixtures. The fluid-dynamic parameters of Reynolds number and transmembrane pressure were varied in the process. The volume of permeate was analyzed by measuring the Total Organic Carbon concentration (TOC), which indicated 99% of oil phase retention. The emulsified mixture was characterized by optical microscopy, while the morphology and composition of the impregnated microporous tubes were analyzed by scanning electron microscopy (SEM). Quantification of the TOC values for the tube impregnated once at 600 degrees C showed the best demulsification performance, with the concentration on permeate smaller than 10 mg/L. The impregnated tube sintered once at 900 degrees C presented low carbon concentration (smaller than 20 mg/L), has the advantage of presenting the greatest trans-membrane flux in relation to the other microporous tube. (c) 2006 Elsevier B.V. All rights reserved.

Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors

Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X(7) receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X(7) receptor. Accordingly, ATP-i cells did not display any detectable ATP-induced current under whole-cell patch-clamp recordings. Using immunofluorescence microscopy, Cx43 reactivity was found at the cell surface and in regions of cell-cell contact of ATP-s cells, whereas, in ATP-i cells, Cx43 immunoreactivity was only present in cytosolic compartments. Using confocal microscopy, it is shown here that, in ATP-s cells as well as in peritoneal macrophages, Cx43 and P2X(7) receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide 0 side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD 147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Location of the β-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022

During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not β-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-β-D-galactopyranoside (ONPG), indicating that β-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that β-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncouplercarbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of β-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed. © 1996 Kluwer Academic Publishers.

Can trifluoperazine protect mitochondria against reactive oxygen species-induced damage?

Trifluoperazine (TFP) (35 μM) prevents mitochondrial transmembrane potential (ΔΨ) collapse and swelling induced by 10 μM Ca2+ plus oxyradicals generated from δ-aminolevulinic acid autoxidation. In contrast with EGTA, TFP cannot restore the totally collapsed ΔΨ. So, TFP might not remove Ca2+ from its 'harmful site', but could impair the ROS-driven cross-linking between membrane -SH proteins. Our data are correlated with the protective uses of TFP against oxidative processes promoted by oxyradicals plus Ca2+.

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.