Dietary supplementation with a natural carotenoid mixture decreases oxidative stress

Objective: To determine whether dietary supplementation with a natural carotenoid mixture counteracts the enhancement of oxidative stress induced by consumption of fish oil. Design: A randomised double-blind crossover dietary intervention. Setting: Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights PO Box 226, Reading RG6 6AP, UK. Subjects and intervention: A total of 32 free-living healthy nonsmoking volunteers were recruited by posters and e-mails in The University of Reading. One volunteer withdrew during the study. The volunteers consumed a daily supplement comprising capsules containing fish oil (4 x 1 g) or fish oil (4 x 1 g) containing a natural carotenoid mixture (4 x 7.6 mg) for 3 weeks in a randomised crossover design separated by a 12 week washout phase. The carotenoid mixture provided a daily intake of beta-carotene (6.0 mg), alpha-carotene (1.4 mg), lycopene (4.5 mg), bixin (11.7 mg), lutein (4.4 mg) and paprika carotenoids (2.2 mg). Blood and urine samples were collected on days 0 and 21 of each dietary period. Results: The carotenoid mixture reduced the fall in ex vivo oxidative stability of low-density lipoprotein (LDL) induced by the fish oil (P = 0.045) and it reduced the extent of DNA damage assessed by the concentration of 8-hydroxy-2'-deoxyguanosine in urine (P = 0.005). There was no effect on the oxidative stability of plasma ex vivo assessed by the oxygen radical absorbance capacity test. beta- Carotene, alpha-carotene, lycopene and lutein were increased in the plasma of subjects consuming the carotenoid mixture. Plasma triglyceride levels were reduced significantly more than the reduction for the fish oil control (P = 0.035), but total cholesterol, HDL and LDL levels were not significantly changed by the consumption of the carotenoid mixture. Conclusions: Consumption of the natural carotenoid mixture lowered the increase in oxidative stress induced by the fish oil as assessed by ex vivo oxidative stability of LDL and DNA degradation product in urine. The carotenoid mixture also enhanced the plasma triglyceride-lowering effect of the fish oil.

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by alpha-(1,2)- and alpha-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases alpha-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for alpha-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

Endothelin-converting enzyme-1 degrades internalized somatostatin-14

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

The plasticity of the mirror system: how reward learning modulates cortical motor simulation of others

Cortical motor simulation supports the understanding of others' actions and intentions. This mechanism is thought to rely on the mirror neuron system (MNS), a brain network that is active both during action execution and observation. Indirect evidence suggests that alpha/beta suppression, an electroencephalographic (EEG) index of MNS activity, is modulated by reward. In this study we aimed to test the plasticity of the MNS by directly investigating the link between alpha/beta suppression and reward. 40 individuals from a general population sample took part in an evaluative conditioning experiment, where different neutral faces were associated with high or low reward values. In the test phase, EEG was recorded while participants viewed videoclips of happy expressions made by the conditioned faces. Alpha/beta suppression (identified using event-related desynchronisation of specific independent components) in response to rewarding faces was found to be greater than for non-rewarding faces. This result provides a mechanistic insight into the plasticity of the MNS and, more generally, into the role of reward in modulating physiological responses linked to empathy.

Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.

The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.

Expression of protein kinase C isoforms during cardiac ventricular development.

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.

Imprints of fast-rotating massive stars in the Galactic Bulge

The first stars that formed after the Big Bang were probably massive(1), and they provided the Universe with the first elements heavier than helium (`metals`), which were incorporated into low-mass stars that have survived to the present(2,3). Eight stars in the oldest globular cluster in the Galaxy, NGC 6522, were found to have surface abundances consistent with the gas from which they formed being enriched by massive stars(4) (that is, with higher alpha-element/Fe and Eu/Fe ratios than those of the Sun). However, the same stars have anomalously high abundances of Ba and La with respect to Fe(4), which usually arises through nucleosynthesis in low-mass stars(5) (via the slow-neutron-capture process, or s-process). Recent theory suggests that metal-poor fast-rotating massive stars are able to boost the s-process yields by up to four orders of magnitude(6), which might provide a solution to this contradiction. Here we report a reanalysis of the earlier spectra, which reveals that Y and Sr are also over-abundant with respect to Fe, showing a large scatter similar to that observed in extremely metal-poor stars(7), whereas C abundances are not enhanced. This pattern is best explained as originating in metal-poor fast-rotating massive stars, which might point to a common property of the first stellar generations and even of the `first stars`.

THE ROLE OF DIFFUSIVITY QUENCHING IN FLUX-TRANSPORT DYNAMO MODELS

In the nonlinear phase of a dynamo process, the back-reaction of the magnetic field upon the turbulent motion results in a decrease of the turbulence level and therefore in a suppression of both the magnetic field amplification (the alpha-quenching effect) and the turbulent magnetic diffusivity (the eta-quenching effect). While the former has been widely explored, the effects of eta-quenching in the magnetic field evolution have rarely been considered. In this work, we investigate the role of the suppression of diffusivity in a flux-transport solar dynamo model that also includes a nonlinear alpha-quenching term. Our results indicate that, although for alpha-quenching the dependence of the magnetic field amplification with the quenching factor is nearly linear, the magnetic field response to eta-quenching is nonlinear and spatially nonuniform. We have found that the magnetic field can be locally amplified in this case, forming long-lived structures whose maximum amplitude can be up to similar to 2.5 times larger at the tachocline and up to similar to 2 times larger at the center of the convection zone than in models without quenching. However, this amplification leads to unobservable effects and to a worse distribution of the magnetic field in the butterfly diagram. Since the dynamo cycle period increases when the efficiency of the quenching increases, we have also explored whether the eta-quenching can cause a diffusion-dominated model to drift into an advection-dominated regime. We have found that models undergoing a large suppression in eta produce a strong segregation of magnetic fields that may lead to unsteady dynamo-oscillations. On the other hand, an initially diffusion-dominated model undergoing a small suppression in eta remains in the diffusion-dominated regime.

A simplified approach for Chaplygin-type cosmologies

A new class of accelerating cosmological models driven by a one-parameter version of the general Chaplygin-type equation of state is proposed. The simplified version is naturally obtained from causality considerations with basis on the adiabatic sound speed vs plus the observed accelerating stage of the universe. We show that very stringent constraints on the unique free parameter a describing the simplified Chaplygin model can be obtained from a joint analysis involving the latest SNe type la data and the recent Sloan Digital Sky Survey measurement of baryon acoustic oscillations (BAO). In our analysis we have considered separately the SNe type la gold sample measured by [A.G. Riess et al.. Astrophys. J. 607 (2004) 665] and the supernova legacy survey (SNLS) from [P. Astier et al., Astron. Astrophys. 447 (2006) 31]. At 95.4% (c.l.), we find for BAO + gold sample, 0.91 <= alpha <= 1.0 and Omega(m) = 0.28(-0.048)(+0.043) while BAO + SNLS analysis provides 0.94 <= alpha <= 1.0 and Omega(m) = 0.27(-0.045)(+0.048). (C) 2008 Elsevier B.V. All rights reserved.

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for alpha-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (alpha, beta, gamma, and delta) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.

Muscle Protein Alterations in LGMD21 Patients With Different Mutations in the Fukutin-related Protein Gene

Fukutin-related protein (FKRP) is a protein involved in the glycosylation of cell surface molecules. Pathogenic mutations in the FKRP gene cause both the more severe congenital muscular dystrophy Type 1C and the milder Limb-Girdle Type 21 form (LGMD21). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, alpha-DG, and alpha 2-laminin, in muscle biopsies from 13 unrelated LGMD21 patients with 10 different FKRP mutations. In all, a typical dystrophic pattern was observed. In eight patients, a high frequency of rimmed vacuoles was also found. A variable degree of alpha 2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented a-DG deficiency on sarcolemmal membranes. Additionally, through Western blot analysis, deficiency of calpain 3 and dystrophin bands was found in four and two patients, respectively. All the remaining proteins showed a similar pattern to normal controls. These results suggest that, in our population of LGMD21 patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of alpha 2-laminin and alpha-DG on sections are prevalent, independently of mutation type or clinical severity.

Basic aminopeptidase activity is an emerging biomarker in collagen-induced rheumatoid arthritis

The objective of this study was to investigate the catalytic activity of basic aminopeptidase (APB) and its association with periarticular edema and circulating tumor necrosis factor (TNF)-alpha and type II collagen (CII) antibodies (AACII) in a rat model of rheumatoid arthritis (RA) induced by CII (CIA). Edema does not occur in part of CH-treated, even when AACII is higher than in control. TNF-alpha is detectable only in edematous CII-treated. APB in synovial membrane is predominantly a membrane-bound activity also present in soluble form and with higher activity in edematous than in non-edematous CH-treated or control. Synovial fluid and blood plasma have lower APB in non-edematous than in edematous CII-treated or control. In peripheral blood mononuclear cells (PBMCs) the highest levels of APB are found in soluble form in control and in membrane-bound form in non-edematous CII-treated. CII treatment distinguishes two categories of rats: one with arthritic edema, high AACII, detectable TNF-alpha, high soluble and membrane-bound APB in synovial membrane and low APB in the soluble fraction of PBMCs, and another without edema and with high AACII, undetectable TNF-alpha, low APB in the synovial fluid and blood plasma and high APB in the membrane-bound fraction of PBMCs. Data suggest that APB and CIA are strongly related. (C) 2011 Elsevier B.V. All rights reserved.

Exercise changes regional vascular control by commissural NTS in spontaneously hypertensive rats

Ogihara CA, Schoorlemmer GHM, Levada AC, Pithon-Curi TC, Curi R, Lopes OU, Colombari E, Sato MA. Exercise changes regional vascular control by commissural NTS in spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 299: R291-R297, 2010. First published April 21, 2010; doi: 10.1152/ajpregu.00055.2009.-Inhibition of the commissural nucleus of the solitary tract (commNTS) induces a fall in sympathetic nerve activity and blood pressure in spontaneously hypertensive rats (SHR), which suggests that this subnucleus of the NTS is a source of sympathoexcitation. Exercise training reduces sympathetic activity and arterial pressure. The purpose of the present study was to investigate whether the swimming exercise can modify the regional vascular responses evoked by inhibition of the commNTS neurons in SHR and normotensive Wistar-Kyoto (WKY) rats. Exercise consisted of swimming, 1 h/day, 5 days/wk for 6 wks, with a load of 2% of the body weight. The day after the last exercise session, the rats were anesthetized with intravenous alpha-chloralose, tracheostomized, and artificially ventilated. The femoral artery was cannulated for mean arterial pressure (MAP) and heart rate recordings, and Doppler flow probes were placed around the lower abdominal aorta and superior mesenteric artery. Microinjection of 50 mM GABA into the commNTS caused similar reductions in MAP in swimming and sedentary SHR (-25 +/- 6 and -30 +/- 5 mmHg, respectively), but hindlimb vascular conductance increased twofold in exercised vs. sedentary SHR (54 +/- 8 vs. 24 +/- 5%). GABA into the commNTS caused smaller reductions in MAP in swimming and sedentary WKY rats (-20 +/- 4 and -16 +/- 2 mmHg). Hindlimb conductance increased fourfold in exercised vs. sedentary WKY rats (75 +/- 2% vs. 19 +/- 3%). Therefore, our data suggest that the swimming exercise induced changes in commNTS neurons, as shown by a greater enhancement of hindlimb vasodilatation in WKY vs. SHR rats in response to GABAergic inhibition of these neurons.

Intracellular peptides as natural regulators of cell signaling

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 mu M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R-CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.