Phosphate Regulated Proteins Of Xanthomonas Citri Subsp. Citri: A Proteomic Approach.

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.

The Soluble Guanylyl Cyclase Activator Bay 60-2770 Potently Relaxes The Pulmonary Artery On Congenital Diaphragmatic Hernia Rabbit Model.

Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage, and a significant cause of morbidity and mortality. In this study, we have used a rabbit model of CDH to evaluate the effects of BAY 60-2770 on the in vitro reactivity of left pulmonary artery. CDH was performed in New Zealand rabbit fetuses (n = 10 per group) and compared to controls. Measurements of body, total and left lung weights (BW, TLW, LLW) were done. Pulmonary artery rings were pre-contracted with phenylephrine (10 μM), after which cumulative concentration-response curves to glyceryl trinitrate (GTN; NO donor), tadalafil (PDE5 inhibitor) and BAY 60-2770 (sGC activator) were obtained as well as the levels of NO (NO3/NO2). LLW, TLW and LBR were decreased in CDH (p < 0.05). In left pulmonary artery, the potency (pEC50) for GTN was markedly lower in CDH (8.25 ± 0.02 versus 9.27 ± 0.03; p < 0.01). In contrast, the potency for BAY 60-2770 was markedly greater in CDH (11.7 ± 0.03 versus 10.5 ± 0.06; p < 0.01). The NO2/NO3 levels were 62 % higher in CDH (p < 0.05). BAY 60-2770 exhibits a greater potency to relax the pulmonary artery in CDH, indicating a potential use for pulmonary hypertension in this disease.

Bay 41-2272, A Soluble Guanylate Cyclase Stimulator, Relaxes Isolated Human Ureter In A Standardized In Vitro Model.

To characterize the relaxation induced by BAY 41-2272 in human ureteral segments. Ureter specimens (n = 17) from multiple organ human deceased donors (mean age 40 ± 3.2 years, male/female ratio 2:1) were used to characterize the relaxing response of BAY 41-2272. Immunohistochemical analysis for endothelial and neuronal nitric oxide synthase, guanylate cyclase stimulator (sGC) and type 5 phosphodiesterase was also performed. The potency values were determined as the negative log of the molar to produce 50% of the maximal relaxation in potassium chloride-precontracted specimens. The unpaired Student t test was used for the comparisons. Immunohistochemistry revealed the presence of endothelial nitric oxide synthase in vessel endothelia and neuronal nitric oxide synthase in urothelium and nerve structures. sGC was expressed in the smooth muscle and urothelium layer, and type 5 phosphodiesterase was present in the smooth muscle only. BAY 41-2272 (0.001-100 μM) relaxed the isolated ureter in a concentration dependent manner, with a potency and maximal relaxation value of 5.82 ± 0.14 and 84% ± 5%, respectively. The addition of nitric oxide synthase and sGC inhibitors reduced the maximal relaxation values by 21% and 45%, respectively. However, the presence of sildenafil (100 nM) significantly potentiated (6.47 ± 0.10, P <.05) this response. Neither glibenclamide or tetraethylammonium nor ureteral urothelium removal influenced the relaxation response by BAY 41-2272. BAY 41-2272 relaxes the human isolated ureter in a concentration-dependent manner, mainly by activating the sGC enzyme in smooth muscle cells rather than in the urothelium, although a cyclic guanosine monophosphate-independent mechanism might have a role. The potassium channels do not seem to be involved.

Inactivation Of Ampk Mediates High Phosphate-induced Extracellular Matrix Accumulation Via Nox4/tgfß-1 Signaling In Human Mesangial Cells.

High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.

Oligomerization, Membrane Association, And In Vivo Phosphorylation Of Sugarcane Udp-glucose Pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

Immobilization Of Papain On Chitin And Chitosan And Recycling Of Soluble Enzyme For Deflocculation Of Saccharomyces Cerevisiae From Bioethanol Distilleries.

Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1-10% w·v(-1)), polyethyleneimine (0.5% v·v(-1)), and tripolyphosphate (1-10% w·v(-1)) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L(-1). Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Pharmacological Characterization Of The Relaxation Induced By The Soluble Guanylate Cyclase Activator, Bay 60-2770 In Rabbit Corpus Cavernosum.

To characterize the relaxation induced by the soluble guanylate cyclase (sGC) activator, BAY 60-2770 in rabbit corpus cavernosum. Penis from male New Zealand rabbits were removed and fours strips of corpus cavernosum (CC) were obtained. Concentration-response curves to BAY 60-2770 were carried out in the absence and presence of inhibitors of nitric oxide synthase, L-NAME (100 μM), sGC, ODQ (10 μM) and phosphodiestarase type 5, tadalafil (0.1 μM). The potency (pEC50) and maximal response (Emax) values were determined. Second, electrical-field stimulation (EFS)-induced contraction or relaxation was realized in the absence and presence of BAY 60-2770 (0.1 or 1 μM) alone or in combination of ODQ (10 μM). In the case of EFS-induced relaxation two protocols were realized: 1) ODQ (10 μM) was first incubated for 20 min and then BAY 60-2770 (1 μM) was added for another 20 min (ODQ + BAY 60-2770). In different CC strips, BAY 60-2770 was incubated for 20 min followed by another 20 min with ODQ (BAY 60-2770 + ODQ). The intracellular levels of cyclic guanosine monophosphate (cGMP) were also determined. BAY 60-2770 potently relaxed rabbit CC with pEC50 and Emax values of 7.58 ± 0.19 and 81 ± 4%, respectively. The inhibitors ODQ (n=7) or tadalafil (n=7) produced 4.2- and 6.3-leftward shifts, respectively in BAY 60-2770-induced relaxation without interfering on the Emax values. The intracellular levels of cGMP were augmented after stimulation with BAY 60-2770 (1 μM) alone, whereas its co-incubation with ODQ produced even higher levels of cGMP. The EFS-induced contraction was reduced in the presence of BAY 60-2770 (1 μM) and this inhibition was even greater when BAY 60-2770 was co-incubated with ODQ. The nitrergic stimulation induced CC relaxation, which was abolished in the presence of ODQ. BAY 60-2770 alone increased the amplitude of relaxation. Co-incubation of ODQ and BAY 60-2770 did not alter the relaxation in comparison with ODQ alone. Interestingly, when BAY 60-2770 was incubated prior to ODQ, EFS-induced relaxation was partly restored in comparison with ODQ alone or ODQ + BAY 60-2770. Considering that the relaxation induced by the sGC activator, BAY 60-2770 was increased after sGC oxidation and unaltered in the absence of nitric oxide, these class of substances are advantageous over sGC stimulators or PDE5 inhibitors for the treatment in those patients with erectile dysfunction and high endothelial damage. This article is protected by copyright. All rights reserved.

Aplicação de métodos de calibração multivariada para a determinação simultânea de riboflavina (VB2), tiamina (VB1), piridoxina (VB6) e nicotinamida (VPP)

In this work, the artificial neural networks (ANN) and partial least squares (PLS) regression were applied to UV spectral data for quantitative determination of thiamin hydrochloride (VB1), riboflavin phosphate (VB2), pyridoxine hydrochloride (VB6) and nicotinamide (VPP) in pharmaceutical samples. For calibration purposes, commercial samples in 0.2 mol L-1 acetate buffer (pH 4.0) were employed as standards. The concentration ranges used in the calibration step were: 0.1 - 7.5 mg L-1 for VB1, 0.1 - 3.0 mg L-1 for VB2, 0.1 - 3.0 mg L-1 for VB6 and 0.4 - 30.0 mg L-1 for VPP. From the results it is possible to verify that both methods can be successfully applied for these determinations. The similar error values were obtained by using neural network or PLS methods. The proposed methodology is simple, rapid and can be easily used in quality control laboratories.

Estudo por espectroscopia micro-Raman dos mecanismos de separação de fase em vidros fosfatos de metais de transição

Glass-ceramics are prepared by controlled separation of crystal phases in glasses, leading to uniform and dense grain structures. On the other hand, chemical leaching of soluble crystal phases yields porous glass-ceramics with important applications. Here, glass/ceramic interfaces of niobo-, vanado- and titano-phosphate glasses were studied by micro-Raman spectroscopy, whose spatial resolution revealed the multiphase structures. Phase-separation mechanisms were also determined by this technique, revealing that interface composition remained unchanged as the crystallization front advanced for niobo- and vanadophosphate glasses (interface-controlled crystallization). For titanophosphate glasses, phase composition changed continuously with time up to the equilibrium composition, indicating a spinodal-type phase separation.