Protein-Protein Binding Kinetics by Biolayer Interferometry.

The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.

Structural biology of complement receptors.

The complement system plays crucial roles in a wide breadth of immune and inflammatory processes and is frequently cited as an etiological or aggravating factor in many human diseases, from asthma to cancer. Complement receptors encompass at least eight proteins from four structural classes, orchestrating complement-mediated humoral and cellular effector responses and coordinating the complex cross-talk between innate and adaptive immunity. The progressive increase in understanding of the structural features of the main complement factors, activated proteolytic fragments, and their assemblies have spurred a renewed interest in deciphering their receptor complexes. In this review, we describe what is currently known about the structural biology of the complement receptors and their complexes with natural agonists and pharmacological antagonists. We highlight the fundamental concepts and the gray areas where issues and problems have been identified, including current research gaps. We seek to offer guidance into the structural biology of the complement system as structural information underlies fundamental and therapeutic research endeavors. Finally, we also indicate what we believe are potential developments in the field.

The crystal structure of iC3b-CR3 αI reveals a modular recognition of the main opsonin iC3b by the CR3 integrin receptor.

Complement activation on cell surfaces leads to the massive deposition of C3b, iC3b, and C3dg, the main complement opsonins. Recognition of iC3b by complement receptor type 3 (CR3) fosters pathogen opsonophagocytosis by macrophages and the stimulation of adaptive immunity by complement-opsonized antigens. Here, we present the crystallographic structure of the complex between human iC3b and the von Willebrand A inserted domain of the α chain of CR3 (αI). The crystal contains two composite interfaces for CR3 αI, encompassing distinct sets of contiguous macroglobulin (MG) domains on the C3c moiety, MG1-MG2 and MG6-MG7 domains. These composite binding sites define two iC3b-CR3 αI complexes characterized by specific rearrangements of the two semi-independent modules, C3c moiety and TED domain. Furthermore, we show the structure of iC3b in a physiologically-relevant extended conformation. Based on previously available data and novel insights reported herein, we propose an integrative model that reconciles conflicting facts about iC3b structure and function and explains the molecular basis for iC3b selective recognition by CR3 on opsonized surfaces.

Antioxidant and Hepatoprotective Effects of Croton hypoleucus Extract in an Induced-Necrosis Model in Rats.

The aim of this study was to evaluate the antioxidant and hepatoprotective activity of Croton hypoleucus (EC). The present work reports the first pharmacological, toxicological, and antioxidant studies of EC extract on liver injury. Liver necrosis was induced by thioacetamide (TAA). Five groups were established: Croton Extract (EC), thioacetamide (TAA), Croton extract with thioacetamide (EC + TAA), vitamin E with thioacetamide (VE + TAA) and the positive control and vehicle (CT). For EC and EC + TAA, Wistar rats (n = 8) were intragastrically pre-administered for 4 days with EC (300 mg/kg.day) and on the last day, EC + TAA received a single dose of TAA (400 mg/kg). At 24 h after damage induction, animals were sacrificed. In vitro activity and gene expression of superoxide dismutase (SOD), catalase (Cat), and Nrf2 nuclear factor were measured. The results show that EC has medium antioxidant properties, with an IC50 of 0.63 mg/mL and a ferric-reducing power of 279.8 µM/mg. Additionally, EC reduced hepatic damage markers at 24 h after TAA intoxication; also, it increased SOD and Cat gene expression against TAA by controlling antioxidant defense levels. Our findings demonstrated the hepatoprotective effect of EC by reducing hepatic damage markers and controlling antioxidant defense levels. Further studies are necessary to identify the mechanism of this protection.

The Nutritional Components of Beer and Its Relationship with Neurodegeneration and Alzheimer's Disease.

The prevalence of degenerative diseases has risen in western countries. Growing evidence suggests that demenia and other cognition affectations are associated with ambient factors including specific nutrients, food ingredients or specific dietary patterns. Mediterranean diet adherence has been associated with various health benefits and decreased risk of many diseases, including neurodegenerative disorders. Beer, as part of this protective diet, contains compounds such as silicon and hops that could play a major role in preventing brain disorders. In this review, different topics regarding Mediterranean diet, beer and the consumption of their main compounds and their relation to neurological health have been addressed. Taking into account published results from our group and other studies, the hypothesis linking aluminum intoxication with dementia and/or Alzheimer's disease and the potential role of regular beer has also been considered. Beer, in spite of its alcohol content, may have some health benefits; nonetheless, its consumption is not adequate for all subjects. Thus, this review analyzed some promising results of non-alcoholic beer on several mechanisms engaged in neurodegeneration such as inflammation, oxidation, and cholinesterase activity, and their contribution to the behavioral modifications induced by aluminum intoxication. The review ends by giving conclusions and suggesting future topics of research related to moderate beer consumption and/or the consumption of its major compounds as a potential instrument for protecting against neurodegenerative disease progression and the need to develop nutrigenetic and nutrigenomic studies in aged people and animal models.

Lipoprotein Profile in Aged Rats Fed Chia Oil- or Hydroxytyrosol-Enriched Pork in High Cholesterol/High Saturated Fat Diets.

Restructuring pork (RP) by adding new functional ingredients, like Chia oil (one of the richest natural source of α-linolenic acid) or hydroxytyrosol (HxT) (potent antioxidant), both with hypolipidemic activities, is one of the strategies that may help to reduce the potential negative effects of high meat products consumption. The aim of this study was to evaluate the Chia oil- or HxT-enriched-RP effect on the lipoprotein profile of aged rats fed high-fat, high-energy, and cholesterol-enriched diets. RP samples were prepared by mixing lean pork and lard with or without Chia oil (152.2 g/kg fresh matter) or HxT (3.6 g/kg fresh matter). Diets were prepared by mixing a semisynthetic diet with freeze-dried RP. Groups of 1-year male Wistar rats were fed the following experimental diets for 8 weeks: C, control-RP diet; HC, cholesterol-enriched-RP diet; and Chia oil-RP (CHIA) and HxT, Chia oil- or hydroxytyrosol-RP, cholesterol-enriched diet. Plasma lipid, lipoprotein profile, SREBP-1c protein, and low-density lipoproteins (LDL) receptor gene (Ldlr) expressions were evaluated. Compared to C diet, the HC diet increased plasma cholesterol, triglycerides, free fatty acids, total lipids, and SREBP-1c expression, but reduced Ldlr expression and significantly modified the lipoprotein profile, giving rise to the presence of high levels of atherogenic cholesterol-enriched very low-density lipoproteins (VLDL) particles. Compared to the HC diet, the HxT diet did not produce significant changes in feed intake but it reduced the body weight. Chia oil and HxT partially arrested the negative effects of the high-fat, high-energy, and cholesterol-enriched meat-based diets on lipemia and lipoproteinemia, mostly by reducing the amount of cholesterol content in VLDL (60% and 74% less in CHIA and HxT vs. HC, respectively) and the VLDL total mass (59% and 63% less in CHIA and HxT vs. HC, respectively). Free fatty acids (FFA) significantly correlated with adipose tissue weight and VLDL total mass (both p < 0.05), and plasma triglycerides, phospholipids, total lipids, and SREBP-1c (all p < 0.001), suggesting the important role of FFA in lipoprotein metabolism. Results support the recommendation to include these ingredients in pork products addressed to reduce the presence of increased atherogenic particles in aged people at CVD risk consuming large amounts of pork.

Silicon Alleviates Nonalcoholic Steatohepatitis by Reducing Apoptosis in Aged Wistar Rats Fed a High-Saturated Fat, High-Cholesterol Diet.

Background: Lipoapoptosis has been identified as a key event in the progression of nonalcoholic fatty liver disease (NAFLD), and hence, antiapoptotic agents have been recommended as a possible effective treatment for nonalcoholic steatohepatitis (NASH). Silicon, included in meat as a functional ingredient, improves lipoprotein profiles and liver antioxidant defenses in aged rats fed a high-saturated fat, high-cholesterol diet (HSHCD). However, to our knowledge, the antiapoptotic effect of this potential functional meat on the liver has never been tested.Objective: This study was designed to evaluate the effect of silicon on NASH development and the potential antiapoptotic properties of silicon in aged rats.Methods: One-year-old male Wistar rats weighing ∼500 g were fed 3 experimental diets containing restructured pork (RP) for 8 wk: 1) a high-saturated fat diet, as an NAFLD control, with 16.9% total fat, 0.14 g cholesterol/kg diet, and 46.8 mg SiO2/kg (control); 2) the HSHCD as a model of NASH, with 16.6% total fat, 16.3 g cholesterol/kg diet, and 46.8 mg SiO2/kg [high-cholesterol diet (Chol-C)]; and 3) the HSHCD with silicon-supplemented RP with amounts of fat and cholesterol identical to those in the Chol-C diet, but with 750 mg SiO2/kg (Chol-Si). Detailed histopathological assessments were performed, and the NAFLD activity score (NAS) was calculated. Liver apoptosis and damage markers were evaluated by Western blotting and immunohistochemical staining.Results: Chol-C rats had a higher mean NAS (7.4) than did control rats (1.9; P < 0.001). The score in Chol-Si rats (5.4) was intermediate and different from that in both other groups (P < 0.05). Several liver apoptosis markers-including hepatocyte terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling, cytosolic cytochrome c, apoptosis-inducing factor, caspases 9 and 3, and the mitochondrial Bcl-2-associated X protein (BAX)-to-B-cell lymphoma 2 (BCL2) ratio-were 9-45% lower in Chol-Si than in Chol-C rats (P < 0.05) and did not differ from values in the control group.Conclusions: Supplemental silicon substantially affects NASH development in aged male Wistar rats fed an HSHCD by partially blocking apoptosis. These results suggest that silicon-enriched RP could be used as an effective nutritional strategy in preventing NASH.

Chia Oil-Enriched Restructured Pork Effects on Oxidative and Inflammatory Status of Aged Rats Fed High Cholesterol/High Fat Diets.

Chia oil has the highest recognized α-linolenic acid (ALA) content. ALA is associated with beneficial changes in plasma lipids and the prevention of cardiovascular diseases. Present article aims to analyze the effect of Chia oil-enriched restructured pork (RP) on aged rats in a nonalcoholic steatohepatitis (NASH) model. Groups of six male Wistar rats (1-year old) were fed the experimental diets: control RP diet (C) noncholesterol high saturated; cholesterol-enriched high-saturated fat/high-cholesterol control RP diet (HC) with added cholesterol and cholic acid; and Chia oil- or Hydroxytyrosol RP cholesterol-enriched high-saturated fat/high cholesterol (CHIA and HxT). Total cholesterol, hepatosomatic index, Nrf2, antioxidant, and inflammation markers were determined. CHIA reduced the hypercholesterolemic effect by lowering levels similar to C; also, ameliorated redox index. CHIA, despite high polyunsaturated fatty acids (PUFA) content, reduced thiobarbituric acid reactive substances (TBARS) and induced the lowest SOD protein synthesis but not a reduction on its activity. Chia oil activated the Nrf2 to arrest the pro-oxidative response to cholesterol and aging. Endothelial nitric oxide synthase (eNOS) system was lower in HxT than in CHIA, suggesting its antiatherogenic activity and related protective effect against high PUFA. Increase in tumor necrosis factor alpha (TNFα) was partially blocked by CHIA. Chia oil has the ability to prevent oxidative damage and modify the inflammatory response, suggesting adequate regulation of the antioxidant system. Results stress the importance of incorporating ALA into the diet.

Glucomannan- and glucomannan plus spirulina-enriched pork affect liver fatty acid profile, LDL receptor expression and antioxidant status in Zucker fa/fa rats fed atherogenic diets.

We evaluated the effects of glucomannan or glucomannan plus spirulina-restructured pork (RP) on liver fatty acid profile, desaturase/elongase enzyme activities and oxidative status of Zucker fa/fa rats for seven weeks. Control (C), glucomannan (G) and glucomannan/spirulina (GS)-RP; HC (cholesterol-enriched control), HG and HGS (cholesterol-enriched glucomannan and glucomannan/spirulina-RP) experimental diets were tested. Increased metabolic syndrome markers were found in C, G and GS rats. Cholesterol feeding increased liver size, fat, and cholesterol and reduced antioxidant enzyme levels and expressions. Cholesterolemia was lower in HG and HGS than in HC. GS vs. G showed higher stearic but lower oleic levels. SFA and PUFA decreased while MUFA increased by cholesterol feeding. The arachidonic/linoleic and docosahexaenoic/alpha-linolenic ratios were lower in HC, HG, and HGS vs. C, G, and GS, respectively, suggesting a delta-6-elongase-desaturase system inhibition. Moreover, cholesterol feeding, mainly in HGS, decreased low-density-lipoprotein receptor expression and the delta-5-desaturase activity and increased the delta-9-desaturase activity. In conclusion, the liver production of highly unsaturated fatty acids was limited to decrease their oxidation in presence of hypercholesterolaemia. Glucomannan or glucomannan/spirulina-RP has added new attributes to their functional properties in meat, partially arresting the negative effects induced by high-fat-high-cholesterol feeding on the liver fatty acid and antioxidant statuses.

Effects of Silicon vs. Hydroxytyrosol-Enriched Restructured Pork on Liver Oxidation Status of Aged Rats Fed High-Saturated/High-Cholesterol Diets.

BACKGROUND: Pork is an essential component of the diet that has been linked with major degenerative diseases and development of non-alcoholic steatohepatitis (NASH). Previous studies have. Previous studies have demonstrated the in vitro antioxidant activity of silicon (Si). Furthermore, when Si is added to restructured pork (RP) strongly counterbalances the negative effect of high-cholesterol-ingestion, acting as an active hypocholesterolemic and hypolipemic dietary ingredient in aged rats. OBJECTIVE: This study was designed to evaluate the effects of Si vs hydroxytyrosol (HxT) RP on liver antioxidant defense in aged rats fed cholesterol-enriched high saturated/high cholesterol diets as a NASH model. METHODS: Four diets were prepared: Control RP diet (C) with non-added cholesterol; Cholesterol-enriched high-saturated/high-cholesterol control RP diet (CHOL-C) with added cholesterol and cholic acid; Si- or HxT-RP cholesterol-enriched high-saturated/high-cholesterol diets (CHOL-Si and CHOL-HxT). Groups of six male Wistar rats (1-yr old) were fed these modified diets for eight weeks. Total cholesterol, hepatosomatic index, liver Nrf2 and antioxidant (CAT, SOD, GSH, GSSG, GR, GPx) markers were determined. RESULTS: Both CHOL-Si and CHOL-HxT diets enhanced the liver antioxidant status, reduced hepatosomatic index and increased SOD actvity. Hydrogen peroxide removal seemed to be involved, explaining that the value of redox index was even lower than C without changing the CAT activity. CHOL-Si results were quite better than CHOL-HxT in most measured parameters. CONCLUSIONS: Our study suggests that Si incorporated into RP matrix was able to counterbalance, more efficiently than HxT, the deleterious effect of consuming a high-saturated/high-cholesterol diet, by improving the liver antioxidant defenses in the context of NASH.

Silicon-Enriched Restructured Pork Affects the Lipoprotein Profile, VLDL Oxidation, and LDL Receptor Gene Expression in Aged Rats Fed an Atherogenic Diet.

BACKGROUND: Research has shown that silicon can play an important role in protecting against degenerative diseases. Restructuring pork by partially disassembling meat permits the incorporation of active components with potential functional effects. However, there has been no research to date on the impact that silicon, as a functional ingredient in restructured pork (RP), has on lipoprotein composition, metabolism, and oxidation. OBJECTIVE: This study was designed to evaluate the effect of silicon-enriched RP on lipemia, lipoprotein profile, and oxidation markers of aged rats fed high-fat, high-energy, cholesterol-enriched diets. METHODS: RP samples similar to commercial sausages (16% protein and 22% fat, wt:wt) were prepared by mixing lean pork and lard alone or with silicon (1.3 g Si/kg fresh matter) under controlled conditions and then freeze-dried. Saturated fat-rich diets were designed by mixing 78.3% purified diet with 21.7% freeze-dried RP. Three groups composed of 8 aged male Wistar rats (1 y old) were fed for 8 wk a control RP (C) diet, a cholesterol-enriched RP (Chol-C) diet [C diet enriched with 1.26% cholesterol plus 0.25% cholic acid, or a cholesterol and silicon-enriched RP (Chol-Si) diet (same as the Chol-C diet but containing silicon)]. Plasma lipid concentrations, lipoprotein profile, the degree of VLDL oxidation, and LDL receptor gene (Ldlr) expression were tested. RESULTS: Compared with the C diet, the Chol-C diet did not modify food intake or body weight but significantly increased (P < 0.05) plasma cholesterol (32%) and total lipids (19%), VLDL and intermediate density lipoprotein + LDL cholesterol (both >600%), total lipids and proteins (both >300%), and the degree of VLDL oxidation [conjugated dienes >250%; thiobarbituric acid-reactive substance (TBARS), 900%] and reduced Ldlr expression (64%) and liver arylesterase activity (54%). The Chol-Si diet partially normalized changes induced by the Chol-C diet. Compared with the Chol-C group, Chol-Si rats had lower VLDL compound concentrations (P < 0.001; e.g., 75% less VLDL cholesterol) and VLDL oxidation (65% less conjugated dienes and 85% less TBARS) but greater Ldlr expression (200%). CONCLUSIONS: Silicon added to RP strongly counterbalanced the negative effect of high-cholesterol-ingestion, functioning as an active hypocholesterolemic, hypolipemic, and antioxidative dietary ingredient in aged rats.

Silicon as neuroprotector or neurotoxic in the human neuroblastoma SH-SY5Y cell line.

Silicon (Si) is a trace element that has been considered to be an environmental contaminant for many years, although different studies have recently reported it is an essential element for living cells. The present study tested the ability of different concentrations of Si G57™ to induce neuroprotection or neurotoxicity over 24 h in the SH-SY5Y human neuroblastoma cell line. Cell viability, cellular proliferation, LDH release, ROS, antioxidant capacity, TBARS, caspase-3, -8 and -9, DNA fragmentation, and TNF-α levels were evaluated. Low Si doses (50-250 ng mL(-1)) increased the cell viability and reduced caspase-3 and -8 activities and TNF-α level. The increase in cell viability was independent of any proliferative effect as there was no variation in cyclin E and PCNA levels. At higher concentrations, Si increased caspase-3, as well as TBARS, LDH, DNA fragmentation, and TNF-α releases. Altogether, these results suggest that Si could act either as a neuroprotector or a neurotoxic agent depending on the concentration tested. This study emphasizes the importance of developing new neuroprotective therapies based on low Si doses.