Staphylococcus aureus Exfoliative Toxin E,Oligomeric State and Flip of P186: Implications for Its Action Mechanism

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.     

The Generation of Nitric Oxide from Aldehyde Dehydrogenase-2: The Role of Dietary Nitrates and Their Implication in Cardiovascular Disease Management

Reduced bioavailability of the nitric oxide (NO) signaling molecule has been associated with the onset of cardiovascular disease. One of the better-known and effective therapies for cardiovascular disorders is the use of organic nitrates, such as glyceryl trinitrate (GTN), which increases the concentration of NO. Unfortunately, chronic use of this therapy can induce a phenomenon known as “nitrate tolerance”, which is defined as the loss of hemodynamic effects and a reduction in therapeutic effects. As such, a higher dosage of GTN is required in order to achieve the same vasodilatory and antiplatelet effects. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a cardioprotective enzyme that catalyzes the bio-activation of GTN to NO. Nitrate tolerance is accompanied by an increase in oxidative stress, endothelial dysfunction, and sympathetic activation, as well as a loss of the catalytic activity of ALDH2 itself. On the basis of current knowledge, nitrate intake in the diet would guarantee a concentration of NO such as to avoid (or at least reduce) treatment with GTN and the consequent onset of nitrate tolerance in the course of cardiovascular diseases, so as not to make necessary the increase in GTN concentrations and the possible inhibition/alteration of ALDH2, which aggravates the problem of a positive feedback mechanism. Therefore, the purpose of this review is to summarize data relating to the introduction into the diet of some natural products that could assist pharmacological therapy in order to provide the NO necessary to reduce the intake of GTN and the phenomenon of nitrate tolerance and to ensure the correct catalytic activity of ALDH2.     

Effects of different particle deposition parameters on adhesion of resin cement to zirconium dioxide and phase transformation

This study evaluated the effect of air-abrasion parameters such as particle size, distance, and time on adhesion of resin cement to zirconium dioxide (Y-TZP) and t→m phase transformation. Y-TZP blocks (N = 80) (In-Ceram YZ, Vita) (4 mm^3?×?4 mm^3?×?3 mm³) were assigned into eight groups (n = 10): air-abrasion with 30 μm (CoJet Sand, S30) and 110 μm (Rocatec-Plus, S110) silica-coated alumina particles, applied for either for 10–20 s (T = time), from a distance of 10–20 mm (D = distance), composing the following groups: S₃₀T₁₀D₁₀, S₃₀T₁₀D₂₀, S₃₀T₂₀D₁₀, S₃₀T₂₀D₂₀, S₁₁₀T₁₀D₁₀, S₁₁₀T₁₀D₂₀, S₁₁₀T₂₀D₁₀, and S₁₁₀T₂₀D₂₀. Resin composite (RelyX ARC) was bonded to Y-TZP blocks in polyethylene molds. The specimens were aged (10,000 thermal cycles and water storage for 90 days) prior to shear bond test. Failure types were analyzed under stereomicroscope and SEM, and phase transformation was calculated. Data (MPa) were analyzed using 3-way ANOVA and Tukey’s tests. Air-abrasion with 110 μm silica particles (10.96) presented significantly higher bond strength (p = 0.0149) compared to 30 μm (8.96). Time (p = 0.403) and distance (p = 0.179) parameters did not affect the results significantly. Air-abrasion with 110 μm particles (12.3) promoted higher bond strength than that of 30 μm (6.4) when applied for 10 s from a distance of 10 mm (Tukey’s). Failure types were predominantly adhesive. Phase transformation ranged between 30.3 and 35.9% for 30 μm particles and 23.8–43.7% for 110 μm particles. While the size of silica-coated alumina particles were more relevant parameter for resin cement adhesion to Y-TZP, time (up to 20 s) and distance (up to 20 mm) appear to be less pertinent.     

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.     

Programmed Cell Death in the Small Intestine: Implications for the Pathogenesis of Celiac Disease

The small intestine has a high rate of cell turnover under homeostatic conditions, and this increases further in response to infection or damage. Epithelial cells mostly die by apoptosis, but recent studies indicate that this may also involve pro-inflammatory pathways of programmed cell death, such as pyroptosis and necroptosis. Celiac disease (CD), the most prevalent immune-based enteropathy, is caused by loss of oral tolerance to peptides derived from wheat, rye, and barley in genetically predisposed individuals. Although cytotoxic cells and gluten-specific CD4⁺ Th1 cells are the central players in the pathology, inflammatory pathways induced by cell death may participate in driving and sustaining the disease through the release of alarmins. In this review, we summarize the recent literature addressing the role of programmed cell death pathways in the small intestine, describing how these mechanisms may contribute to CD and discussing their potential implications.     

Anti-Listeria monocytogenes activity of enterocins microencapsulated by ionic gelation

The encapsulation of enterocins synthesized by Enterococcus faecium CRL1385 through ionic gelation with calcium ions was analyzed. Different enterocins samples were lyophilised and encapsulated using low-methoxyl pectin as the coating material. Lipids present in milk butter were also added to control the release of antimicrobial peptides from the capsules. The morphology of fresh and freeze-dried capsules was examined using light microscopy and scanning electron microscopy, respectively. Antimicrobial activity of encapsulated bacteriocins was assessed against Listeria monocytogenes 01/155 using the agar diffusion technique and direct contact in microplates. The capsules with higher lipid content showed a more spherical and uniform shape. Pathogen inhibition was observed for capsules prepared with different bacteriocin solutions both on solid (halo diameter = 8.5–13.5 mm) and in an aqueous medium (ca. 2 log orders decline in L. monocytogenes viability). The outcomes suggest that bacteriocin encapsulation through ionic gelation can be a potential alternative for the application of these antimicrobial peptides as biopreservatives in food.     

Use of a new milk-clotting protease from Thermomucor indicae-seudaticae N31 as coagulant and changes during ripening of Prato cheese

Prato cheeses were manufactured using coagulant from Thermomucor indicae-seudaticae N31 or a commercial coagulant. Cheeses were characterised using the following analysis: yield; fat; acidity; moisture; ash; salt; pH; total nitrogen; total protein; NS-pH 4.6/NT*100; NS-TCA 12%/NT*100; casein electrophoresis; and RP-HPLC. The results were statistically analysed and revealed that the proteolytic indices were not significantly different throughout the 60 days of ripening of cheeses made with either coagulant. Even though there were some quantitative differences in the peptide profile of cheeses, the enzyme from T. indicae-seudaticae N31 was used in the production of good quality Prato cheese without having to change the established technological parameters of the process.     

Rats (Rattus norvegicus) in a water maze learn both an egocentric trajectory and landmarks.

In two experiments we investigated the extent to which rats (Rattus norvegicus) use an egocentric trajectory and landmarks to locate a goal. In Experiment 1 we trained groups to locate the hidden platform in a water maze with either 1 of 3 or 3 of 3 predictive landmarks, and with either a random or fixed egocentric trajectory. A choice test revealed that regardless of the landmark configuration, rats relied on a directional, egocentric trajectory, when it was available, to locate the platform. In Experiment 2 we found that adding four predictive landmarks following training with a constant egocentric trajectory did not alter rats' initial attention to the trajectory. We conclude that the presence of nonpredictive landmarks in a predictive array did not affect the use of landmarks. With a blocking design, rats used initially an egocentric path, then landmarks. These results add to the notion that animals use available spatial cues sequentially. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Immunological determination of gliadin 33‐mer equivalent peptides in beers as a specific and practical analytical method to assess safety for celiac patients

BACKGROUND: Cereals used for beer manufacturing contain gluten, which is immunotoxic for celiac patients. The gluten remaining after processes of malting and brewing is mostly hydrolyzed, which makes practical evaluation of the immunotoxicity of the gluten pools challenging. RESULTS: We analyzed the presence of gluten peptides equivalent to the major immunotoxic protease‐resistant gliadin 33‐mer in 100 Belgium beers, using monoclonal antibodies (G12/A1). Immunochromatographic strips and enzyme‐linked immonosorbent assay G12/A1 methods estimated at least 20 ppm gluten equivalents in 90 beers and gluten‐free in 10 beers. The G12/A1 reactivity of beer high‐performance liquid chromatographic fractions correlated to the presence of T‐cell‐reactive epitopes identified by peptide sequencing. CONCLUSION: The determination of equivalent gliadin 33‐mer epitopes in beers has been shown to be practical, specific, and sensitive for the measurement of potential immunotoxicity for celiac patients. Copyright © 2012 Society of Chemical Industry