Influences of the pH on the adsorption properties of an antimicrobial peptide on titanium surfaces

The adsorption behavior of the Tet-124 antimicrobial peptide and the Tet-124 peptide modified at the C- and N-terminus with the sequence glycine-3,4-dihydroxyphenylalanine-glycine (G-DOPA-G) on titanium surfaces was studied using quartz crystal micro balance with dissipation (QCM-D). At a low pH level (4.75) Tet-124 and Tet-124-G-DOPA-G form rigid layers. This is attributed to the electrostatic interactions of the positively charged lysine and arginine residues in the peptide sequence with the negatively charged titanium oxide layer. At an elevated pH level (6.9) Tet-124 shows a lower mass adsorption at the surface than Tet-124-G-DOPA-G. This is attributed to the interaction of the catechol due to the formation of complexes with the titanium oxide and titanium surface layer. The C terminal and N terminal modification with the sequence G-DOPA-G shows similar adsorption rate and mass adsorption coverage at saturation; however it is presented a more loosely layers on the G-DOPA-G-TeT-124. Fibroblast adhesion and the biocompatibility test of both the surfaces following modification with Tet-124-G-DOPA-G and the titanium alloy control showed similar results. In addition, no changes in the adhesion of E. coli bacteria due to the modification of the surface were detected.

Potencial anti-inflamatório e antiproliferativo de compostos inibidores da enzima desoxi-hipusina sintase

Pós-graduação em Biotecnologia - IQ

Modelos para estimar consumo e exigências nutricionais para poedeiras comerciais

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

Avaliação econômica da substituição do farelo de soja por farelo de algodão em dietas práticas para tilápias do Nilo cultivadas em tanques-rede

Based on fish production performance, this study assessed the feasibility of replacing SBM with CSM in practical diets for cage-raised Nile tilapia during the finishing phase. To accomplish this research, four isoproteic (32% CP) and isoenergetic (2500 Kcal/kg of DE) diets were formulated. Control diet (T1) contained 30 % of SBM and 0% of CSM. The other three diets contained: 20% of SBM and 10% of CSM (T2), 10% of SBM and 20% of CSM (T3) and 0% of SBM and 30% of CSM (T4). Lysine and Methionine deficiency was compensated for with synthetic amino acid (L-lysine and DL- methionine) supplementation. Experimental design was completely randomized with four treatments and four repetitions. Data were analyzed with SAS PROC GLM through co-variance analysis, adding initial mean weight as a co-variable, followed by Dunnett test. The system used to calculate the profit part was Partial Budget Analysis in Aquaculture, proposed by Shang (1990). Profit was significantly different at T3 when compared to T1 (control). It was concluded that the addition of 20% of CSM to finishing diets can improve cage-raised Nile tilapia profitability.

Relação ideal dos aminoácidos essenciais para mantença, crescimento e produção de aves

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

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.

Characterization of Glycation Sites on Human Serum Albumin using Mass Spectrometry

The modification of proteins by reducing sugars is a process that occurs naturally in the body. This process, which is known as glycation, has been linked to many of the chronic complications encountered during diabetes. Glycation has also been linked to changes in the binding of human serum albumin (HSA) to several drugs and small solutes in the body. While these effects are known, there is little information that explains why these changes in binding occur. The goal of this project was to obtain qualitative and quantitative information about glycation that occurs on HSA. The first section of this dissertation examined methods that could be used to quantify and identify glycation that occurs on HSA. The extent of glycation that occurred on HSA was quantified using oxygen-18 labeling mass spectrometry and the glycation sites were identified by observing the mass-to-charge (m/z) shifts that occurred in glycated HSA. This initial investigation revealed that oxygen-18 labeling based quantitation can be improved over previous methods if a relative comparison is done with oxygen-18 labeled peptides in a control HSA sample. Similarly, the process of making m/z shift-based assignments could be improved if only the peptides that were unique to the glycated HSA samples were used with internal calibration. These techniques were used in subsequent chapters for the assignment of early and late-stage glycation products on HSA. The regions of HSA that contained the highest amount of modification were identified, quantified, and ranked in order of their relative abundance. Of the commonly reported glycation sites, the N-terminus was found to have the highest extent of modification, followed by lysines 525, 199, and 439. The relative amount of modification on lysine 281, with respect to the aforementioned residues, varied with different degrees of glycation. The oxygen-18 labeling approach used for this analysis was novel because it allowed for the simultaneous quantification of all glycation-related modifications that were occurring on HSA. As such, several arginine residues were also found to have high amounts of modification on glycated HSA.

Cardiovascular effects of the microinjection of L-proline into the third ventricle or the paraventricular nucleus of the hypothalamus in unanesthetized rats

We investigated the cardiovascular effects of the microinjection of L-proline (L-Pro) into the third ventricle (3V) and its peripheral mechanisms. Different doses of L-Pro into the 3V caused dose-related pressor and bradycardiac responses. The pressor response to L-Pro injected into the 3V was potentiated by intravenous pretreatment with the ganglion blocker pentolinium (5 mg/kg), thus excluding any significant involvement of the sympathetic nervous system. Because the response to the microinjection of L-Pro into the 3V was blocked by intravenous pretreatment with the V1-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP (50 mu g/kg), it is suggested that these cardiovascular responses are mediated by a vasopressin release. The pressor response to the microinjection of L-Pro into the 3V was found to be mediated by circulating vasopressin, so, given that the paraventricular nucleus of the hypothalamus (PVN) is readily accessible from the 3V, we investigated whether the PVN could be a site of action for the L-Pro microinjected in the 3V. The microinjection of L-Pro (0.033 mu moles/0.1 mu l) into the PVN caused cardiovascular responses similar to those of injection of the 3V and were also shown to be mediated by vasopressin release. In conclusion, these results show that the microinjection of L-Pro into the 3V causes pressor and bradycardiac responses that could involve stimulation of the magnocellular cells of the PVN and release of vasopressin into the systemic circulation. Also, because the microinjection of L-Pro into the PVN caused a pressor response, this is the first evidence of cardiovascular effects caused by its injection in a supramedullary structure. (c) 2012 Wiley Periodicals, Inc.

Moxonidine into the lateral parabrachial nucleus reduces renal and hormonal responses to cell dehydration

The deactivation of the inhibitory mechanisms with injections of moxonidine (alpha(2)-adrenoceptor/imidazoline receptor agonist) into the lateral parabrachial nucleus (LPBN) increases hypertonic NaCl intake by intra- or extracellular dehydrated rats. In the present study, we investigated the changes in the urinary sodium and volume, sodium balance, and plasma vasopressin and oxytocin in rats treated with intragastric (i.g.) 2 M NaCl load (2 ml/rat) combined with injections of moxonidine into the LPBN. Male Holtzman rats (n=5-12/group) with stainless steel cannulas implanted bilaterally into LPBN were used. Bilateral injections of moxonidine (0.5 nmol/0.2 mu l) into the LPBN decreased i.g. 2 M NaCIinduced diuresis (4.6 +/- 0.7 vs. vehicle: 7.4 +/- 0.6 ml/120 min) and natriuresis (1.65 +/- 0.29 vs. vehicle: 2.53 +/- 0.17 mEq/120 min), whereas the previous injection of the alpha(2)-adrenoceptor antagonist RX 821002 (10 nmol/0.2 mu l) into the LPBN abolished the effects of moxonidline. Moxonidine injected into the LPBN reduced i.g. 2 M NaCl-induced increase in plasma oxytocin and vasopressin (14.6 +/- 2.8 and 2.2 +/- 0.3 vs. vehicle: 25.7 +/- 7 and 4.3 +/- 0.7 pg/ml, respectively). Moxonidine injected into the LPBN combined with i.g. 2 M NaCl also increased 0.3 M NaCl intake (7.5 +/- 1.7 vs. vehicle: 0.5 +/- 0.2 mEq/2 h) and produced positive sodium balance (2.3 +/- 1.4 vs. vehicle: -1.2 +/- 0.4 mEq/2 h) in rats that had access to water and NaCl. The present results show that LPBN alpha(2)-adrenoceptor activation reduces renal and hormonal responses to intracellular dehydration and increases sodium and water intake, which facilitates sodium retention and body fluid volume expansion. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Post-translational modification of the RhoGTPase activating protein 21, ARHGAP21, by SUMO2/3

ARHGAP21 is a 217 kDa RhoGAP protein shown to modulate cell migration through the control of Cdc42 and FAK activities. In the present work a 250 kDa-ARHGAP21 was identified by mass spectrometry. This modified form is differentially expressed among cell lines and human primary cells. Co-immunoprecipitations and in vitro SUMOylation confirmed ARHGAP21 specific modification by SUMO2/3 and mapped the SUMOylation site to ARHGAP21 lysine K1443. Immunofluorescence staining revealed that ARHGAP21 co-localizes with SUMO2/3 in the cytoplasm and membrane compartments. Interestingly, our results suggest that ARHGAP21 SUMOylation may be related to cell proliferation. Therefore, SUMOylation of ARHGAP21 may represent a way of guiding its function. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Physiological effects of glyphosate over amino acid profile in conventional and transgenic soybean (Glycine max)

This paper compares the responses of conventional and transgenic soybean to glyphosate application in terms of the contents of 17 detectable soluble amino acids in leaves, analyzed by HPLC and fluorescence detection. Glutamate, histidine, asparagine, arginine + alanine, glycine + threonine and isoleucine increased in conventional soybean leaves when compared to transgenic soybean leaves, whereas for other amino acids, no significant differences were recorded. Univariate analysis allowed us to make an approximate differentiation between conventional and transgenic lines, observing the changes of some variables by glyphosate application. In addition, by means of the multivariate analysis, using principal components analysis (PCA), cluster analysis (CA) and linear discriminant analysis (LDA) it was possible to identify and discriminate different groups based on the soybean genetic origin. (C) 2011 Elsevier Inc. All rights reserved.

Oxytocin interference in the effects induced by inhalation of 7.5% CO2 in healthy volunteers

Objective The objective of this study was to assess the acute effect of intranasally administered oxytocin (OT) on subjective states, cardiovascular, and endocrine parameters in healthy volunteers who inhaled 7.5% CO2. Methods Forty-five subjects were allocated into three matched groups of subjects who received 24?international units (IU) of OT, 2?mg of lorazepam (LZP), or placebo (PL). The challenge consisted of medical air inhalation for 20?min, 10?min of rest, and CO2 7.5% inhalation for 20?min. Subjective effects were evaluated by self-assessment scales; heart rate, blood pressure, skin conductance, and salivary cortisol were also measured. Assessments were performed at four time points: (i) baseline (-15?min); (ii) post-air inhalation (90?min); (iii) post-CO2 inhalation (120?min), and (iv) post-test (160?min). Results CO2 inhalation significantly increased the anxiety score in the PL group compared with the post-air measurement but not in the OT or LZP groups. The LZP reduced anxiety after medical air inhalation. Other parameters evaluated were not affected by OT. Conclusion OT, as well as LZP, prevented CO2-induced anxiety, suggesting that this hormone has anxiolytic properties. Copyright (C) 2012 John Wiley & Sons, Ltd.

Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes

1. The present study provides the first in vivo evidence that the cannabinoid CB1 receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB1 receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. 2. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB1 receptor. 3. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB1 receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. 4. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB1 receptor in the control of peripheral factors that modulate cardiovascular function. 5. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB1 receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamicpituitaryadrenal axis. 6. Collectively, the results of the present study indicate that the CB1 receptor modulates neurohypophyseal hormone secretion and systemic factors, such as corticosterone and ANP, thus participating in homeostatic responses to altered extracellular volume and plasma tonicity.

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coil, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAD) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut CAD hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C265) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 angstrom, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. (C) 2012 Elsevier Ltd. All rights reserved.

Ractopamine as a Metabolic Modifier Feed Additive for Finishing Pigs: A Review

The beta-adrenergic agonist ractopamine is increasingly used in the swine industry due to higher consumer demand for leaner pork products. Redirecting nutrients to favor leanness rather than fat deposition, ractopamine improves growth and carcass traits of finishing pigs. However, the impact of this agonist on pork quality is not clearly defined. Understanding the biological effects of dietary ractopamine dose, treatment period, lysine levels, and the lysine to metabolizable energy ratio will help pork producers achieve improvements in animal performance, carcass leanness, and economic efficiency in swine production systems.