Effect of polymorphisms in the CD36 and STAT3 genes on different dietary interventions among patients with coronary artery disease: study protocol for a randomized controlled trial

BackgroundCardiovascular disease has become a major health problem, and it has been associated with both environmental and genetic factors. Studies have shown that the Mediterranean Diet (MeDiet), or its components such as nuts and olive oil, may be strongly associated with the improvement of cardiovascular risk factors in specific populations. The purpose of the GENUTRI study is to investigate the interaction of genetics with cardiovascular risk factors in a non-Mediterranean population with coronary artery disease (CAD) according to three different diets: rich in pecan nuts, in extra-virgin olive oil or a control diet.Methods/designThe GENUTRI study is a single-center, randomized, open-label, parallel-group, 12-week pragmatic clinical trial conducted in patients aged 40 to 80 years and diagnosed with CAD. A standardized questionnaire will be applied to data collection and a blood sample will be obtained for lipid, glycemic and inflammatory profile evaluation. Polymorphisms in the CD36 and STAT3 genes will be detected using the TaqMan® SNP Genotyping Assay. Patients will be allocated in three groups: group 1: 30 g/day of pecan nuts; group 2: 30 ml/day of olive oil; and group 3: control diet. The primary outcome will consist of changes in LDL-cholesterol (in mg/dl) after 12 weeks of intervention.DiscussionStudies have shown the beneficial effects of diets rich in nuts and olive oil mainly in the Mediterranean population. GENUTRI is a clinical trial focusing on the effects of nuts or olive oil supplementation in Brazilian individuals. Additionally, we will try to demonstrate that genetic polymorphisms linked to cardiovascular disease may modulate the effects of different diets on biochemical and inflammatory markers among these subjects.

Trial registration

ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT02202265","term_id":"NCT02202265"}}NCT02202265 (registered on 18 July 2014: first version).     

Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2

Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.     

Anti Transglutaminase Antibodies Cause Ataxia in Mice

Background

Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the most common one.

Methods

We analyzed by immunohistochemistry, the anti-neural reactivity of the serum from 20 CD patients. To determine the role of anti-TG2 antibodies in ataxia, two anti-TG2 single chain variable fragments (scFv), isolated from a phage-display IgA antibody library, were characterized by immunohistochemistry and ELISA, and injected in mice to study their effects on motor coordination. We found that 75% of the CD patient population without evidence of neurological involvement, has circulating anti-neural IgA and/or IgG antibodies. Two anti-TG2 scFvs, cloned from one CD patient, stained blood vessels but only one reacted with neurons. This anti-TG2 antibody showed cross reactivity with the transglutaminase isozymes TG3 and TG6. Intraventricular injection of the anti-TG2 or the anti-TG2/3/6 cross-reactive scFv provoked transient, equally intensive ataxia in mice.

Conclusion

The serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia.     

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.     

Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr residue

Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 (⁶⁰⁰SHIFAGGYAAGYYSY⁶¹⁴). Modeling studies indicated that in OpdA the Tyr⁶⁰⁷ residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr⁶⁰⁷ by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. The kinetic parameters showed the importance of Tyr⁶⁰⁷ in OpdA catalytic activity as its substitution promoted a decrease in the k_cat/K_m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr⁶⁰⁷ residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity.     

A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D). Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873. Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube. The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression. Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.     

Metabolic biotinylation as a probe of supramolecular structure of the triad junction in skeletal muscle

Excitation-contraction coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors (DHPRs) in the plasma membrane and ryanodine receptors (RyRs) in the sarcoplasmic reticulum. However, it remains uncertain what regions, if any, of the two proteins interact with one another. Toward this end, it would be valuable to know the spatial interrelationships of DHPRs and RyRs within plasma membrane/sarcoplasmic reticulum junctions. Here we describe a new approach based on metabolic incorporation of biotin into targeted sites of the DHPR. To accomplish this, cDNAs were constructed with a biotin acceptor domain (BAD) fused to selected sites of the DHPR, with fluorescent protein (XFP) attached at a second site. All of the BAD-tagged constructs properly targeted to junctions (as indicted by small puncta of XFP) and were functional for excitation-contraction coupling. To determine whether the introduced BAD was biotinylated and accessible to avidin (approximately 60 kDa), myotubes were fixed, permeablized, and exposed to fluorescently labeled avidin. Upon expression in beta1-null or dysgenic (alpha1S-null) myotubes, punctate avidin fluorescence co-localized with the XFP puncta for BAD attached to the beta1a N- or C-terminals, or the alpha1S N-terminal or II-III loop. However, BAD fused to the alpha1S C-terminal was inaccessible to avidin in dysgenic myotubes (containing RyR1). In contrast, this site was accessible to avidin when the identical construct was expressed in dyspedic myotubes lacking RyR1. These results indicate that avidin has access to a number of sites of the DHPR within fully assembled (RyR1-containing) junctions, but not to the alpha1S C-terminal, which appears to be occluded by the presence of RyR1.     

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.