Flavonas, lignanas e terpeno de Piper umbellata (Piperaceae)

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

Constitutive and Treatment-Induced CXCL8-Signalling Selectively Modulates the Efficacy of Anti-Metabolite Therapeutics in Metastatic Prostate Cancer

Background: The current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent.

Methods: The response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression.

Results: Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P,0.001), and increased 5-FU-induced apoptosis in PC3 cells (P,0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU.

Conclusions: CXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis. © 2012 Wilson et al.

Evaluation of the protective effect of Phlomis purpurea against Phytophthora cinnamomi in Fagaceae and of root metabolites involved

Tese de doutoramento, Ciências Agrárias (Proteção de Plantas), Faculdade de Ciência e Tecnologia, Universidade do Algarve, 2014

Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis. '

Biochemical profiling of proteins and metabolites in wound exudate from chronic wound environments

The lack of fundamental knowledge on the biological processes associated with wound healing represents a significant challenge. Understanding the biochemical changes that occur within a chronic wound could provide insights into the wound environment and enable more effective wound management. We report on the stability of wound fluid samples under various conditions and describe a high-throughput approach to investigate the altered biochemical state within wound samples collected from various types of chronic, ulcerated wounds. Furthermore, we discuss the viability of this approach in the early stages of wound sample protein and metabolite profiling and subsequent biomarker discovery. This approach will facilitate the detection of factors that may correlate with wound severity and/or could be used to monitor the response to a particular treatment.

Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease

Deleterious responses to pathogens during infancy may contribute to infection and associated asthma. Chlamydia respiratory infections in early life are common causes of pneumonia and lead to reduced lung function and asthma. We investigated the role of interleukin-13 (IL-13) in promoting early-life Chlamydia respiratory infection, infection-induced airway hyperresponsiveness (AHR), and severe allergic airway disease (AAD). Infected infant Il13−/− mice had reduced infection, inflammation, and mucus-secreting cell hyperplasia. Surprisingly, infection of wild-type (WT) mice did not increase IL-13 production but reduced IL-13Rα2 decoy receptor levels compared with sham-inoculated controls. Infection of WT but not Il13−/− mice induced persistent AHR. Infection and associated pathology were restored in infected Il13−/− mice by reconstitution with IL-13. Stat6−/− mice were also largely protected. Neutralization of IL-13 during infection prevented subsequent infection-induced severe AAD. Thus, early-life Chlamydia respiratory infection reduces IL-13Rα2 production, which may enhance the effects of constitutive IL-13 and promote more severe infection, persistent AHR, and AAD.

A rapid and sensitive UPLC-ESI MS method for analysis of isofraxidin, a natural antistress compound, and its metabolites in rat plasma

Isofraxidin is one of the main bioactive constituents in the root of Acanthopanax senticosus, which has antifatigue, antistress, and immuno-accommondating effects. In this study, an ultraperformance LC (UPLC)-ESI MS method was developed for analyzing isofraxidin and its metabolites in rat plasma. The analysis was performed on a UPLC coupled with ESI MS (quadropole MS tandem TOF MS). The lower LOD (LLOD) for isofraxidin was 0.25 ng/mL, the intraday precision was less than 10%, the interday precision was less than 10%, and the extraction recovery was more than 80%. Isofraxidin and two metabolites (M1 and M2) were detected in rat plasma after oral administration of isofraxidin, and the molecular polarities of M1 and M2 were both increased compared to isofraxidin. The metabolites were identified as 5,6-dihydroxyl-7-methoxycoumarin and 5-hydroxyl-6,7-dimethoxycoumarin when subjected to parent ion spectra, product ion spectra, and extract mass and element composition analyses.

Hyperelastic constitutive relationship for the strain-rate dependent behavior of shoulder and other joint cartilages

Non-linear finite deformations of articular cartilages under physiological loading conditions can be attributed to hyperelastic behavior. This paper contains experimental results of indentation tests in finite deformation and proposes an empirical based new generalized hyperelastic constitutive model to account for strain-rate dependency for humeral head cartilage tissues. The generalized model is based on existing hyperelastic constitutive relationships that are extensively used to represent biological tissues in biomechanical literature. The experimental results were obtained for three loading velocities, corresponding to low (1x10-3 s-1), moderate and high strain-rates (1x10-1 s-1), which represent physiological loading rates that are experienced in daily activities such as lifting, holding objects and sporting activities. Hyperelastic material parameters were identified by non linear curve fitting procedure. Analysis demonstrated that the material behavior of cartilage can be effectively decoupled into strain-rate independent(elastic) and dependent parts. Further, experiments conducted using different indenters indicated that the parameters obtained are significantly affected by the indenter size, potentially due to structural inhomogeneity of the tissue. The hyperelastic constitutive model developed in this paper opens a new avenue for the exploration of material properties of cartilage tissues.

Nedd4-2functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl– channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 μg/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 μg/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Determination of glutathione transferase (GSTT1-1) activities in different tissues based on formation of radioactive metabolites using 35S-glutathione

A new system has been developed to determine enzyme activities of glutathione transferase θ (GSTT1-1) based on radiometric product detection resulting from the enzymic reaction of methyl chloride with 35S-labelled glutathione. In principle, the method is universally applicable for determination of glutathione transferase activities towards a multiplicity of substrates. The method distinguishes between erythrocyte GSTT1-1 activities of human 'non-conjugators', 'low conjugators' and 'high conjugators'. Application to cytosol preparations of livers and kidneys of male and female Fischer 344 and B6C3F1 mice reveals differential GSTT1-1 activities in hepatic and renal tissues. These ought to be considered in species-specific modellings of organ toxicities of chlorinated hydrocarbons.