Molecular docking simulation analysis of alcohol acyltransferases from two related fruit species explains their different substrate selectivities

Tropical papaya (Carica papaya) and mountain papaya (Vasconcellea pubescens) fruits are characterised for their strong and particular aroma. The aroma of both fruits is different and dominated by esters, which are synthesised by alcohol acyltransferases (AATs). The ability to produce esters is contrasting, V. pubescens (VpAAT1) being a very active enzyme towards the production of benzyl acetate, whereas C. papaya (CpAAT1) is more active towards the production of ethyl butanoate and methyl butanoate, but not benzyl acetate. In order to understand the mechanism of action at the molecular level, the structural model of CpAAT1 protein was built by comparative modelling. Conformational interaction between the protein and several ligands was carried out by molecular docking. CpAAT1 structure showed two domains connected by a large crossover loop, with a solvent channel in the centre of the structure. CpAAT1 and VpAAT1 proteins showed similar 3D structures, including their catalytic sites, but their solvent channels showed differences in size and shape. CpAAT1 solvent channel is larger, in agreement with its higher selectivity for large acyl-CoA substrates. In addition, the most favourably predicted substrate orientation in CpAAT1 was observed for methanol and butanoyl-CoA, showing a perfect coincidence with the high production rate of methyl butanoate of C. papaya fruit.     

Influence of Toxoplasma gondii Acute Infection on Cholinesterase Activities of Wistar Rats

Several studies have shown the mechanisms and importance of immune responses against Toxoplasma gondii infection and the notable role of cholinesterases in inflammatory reactions. However, the association between those factors has not yet been investigated. Therefore, the aim of this study was to evaluate the acetylcholinesterase (AChE) activity in blood and lymphocytes and the activity of butyrylcholinesterase (BChE) in serum of rats experimentally infected with T. gondii during the acute phase of infection. For that, an in vivo study was performed with evaluations of AChE and BChE activities on days 5 and 10 post-infection (PI). The activity of AChE in blood was increased on day 5 PI, while in lymphocytes its activity was enhanced on days 5 and 10 PI (P<0.05). No significant difference was observed between groups regarding to the activity of BChE in serum. A positive (P<0.01) correlation was observed between AChE activity and number of lymphocytes. The role of AChE as an inflammatory marker is well known in different pathologies; thus, our results lead to the hypothesis that AChE has an important role in modulation of early immune responses against T. gondii infection.     

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the K_i in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.     

Bacterial Populations (First Record) at Two Shallow Hydrothermal Vents of the Mexican Pacific West Coast

Thermophilic and metal-oxidizing bacteria were identified in shallow hydrothermal vents on the western Mexican coast. The role of these bacteria in biomineralization processes observed in the vents is explained, and the effect of the vents on biodiversity of prokaryotes is discussed. Research was done at two shallow hydrothermal vent sites: Bahía Concepción (BC) in the Baja California Peninsula and Punta Mita (PM), on the central Pacific coast. Temperature at the sediments proximal to the vents was similar, but the redox potentials (0.5 V in BC and ?0.3 V in PM) and pH (6.2 in BC and as low as 4.5 in PM) differed. The composition of the discharged water ranged from nearly seawater to lower-salinity fluids, and the gas phase was mainly CO₂ at BC and N₂ and CH₄ at PM. The study focuses on the biogeochemical processes related to the different species of bacteria present in the studied sites, which are involved in the anaerobic oxidation of methane (AOM), seawater sulfate reduction, and metal oxidation. The detected bacterial lineages represented typical deep vent species, which disproves a previous hypothesis that proposed that different consortia were populating deep and shallow hydrothermal vents. The results obtained here show that the main parameter affecting the bacterial groups present in shallow vents was the redox potential: gamma-, delta-, and epsilon proteobacteria as well as Bacteriodetes are present under the oxidizing conditions of BC, and Thermotogae, Aquificae, and Planctomycetes are present in PM. Sunlight abundance favored the prevalence of halophilic and Chlorofleaxae bacteria in both areas.     

Human monocyte‐derived macrophages spontaneously differentiated in vitro show distinct phenotypes

Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co‐exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round‐ and spindle‐shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte‐derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)‐10, and transforming growth factor (TGF) β2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C‐C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non‐inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro‐inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions. J. Cell. Physiol. 228: 1464–1472, 2013. © 2012 Wiley Periodicals, Inc.     

EGFR through STAT3 modulates ΔN63α expression to sustain tumor‐initiating cell proliferation in squamous cell carcinomas

Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ΔNp63α isoform. Here, we investigated the regulation of ΔNp63α expression upon EGFR activation and the role of the EGFR–ΔNp63α axis in proliferation of SCC tumor‐initiating cells (TICs). SCC cell lines A‐431, Cal‐27, and SCC‐25 treated with EGF showed a time‐dependent increase in ΔNp63α expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ΔNp63α expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ΔNp63α impaired the TICs ability to grow under non‐differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ΔNp63α accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non‐differentiating conditions. Together, our findings provide evidence that ΔNp63α expression is regulated by EGFR activation through STAT3 and that the EGFR–ΔNp63α axis is crucial for proliferation of TICs present in SCCs. J. Cell. Physiol. 228: 871–878, 2013. © 2012 Wiley Periodicals, Inc.     

Alterations in cerebrospinal fluid glycerophospholipids and phospholipase A2 activity in Alzheimer's disease

Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer''s disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA₂ activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA₂ activity accounts for altered GPs in these fractions as neurodegeneration progresses.