Cholesterol-Lowering Properties of Whole Cowpea Seed and Its Protein Isolate in Hamsters

Hypercholesterolemic hamsters were fed for 4 wk on diets rich in saturated fatty acids and cholesterol, differing only in protein source (20%): casein (control group, HC), whole cowpea seed (HWS), and cowpea protein isolate (HPI). Hamsters fed on HWS and HPI presented significant reductions in plasma total cholesterol and non-HDL cholesterol. HPI and HC presented similar protein digestibility, which were significantly higher than that of HWS. Animals fed on HWS presented significantly higher levels of bile acids and cholesterol in feces than did the animals fed on casein or HPI diets. Histological analyses of the liver showed that HC diet resulted in steatosis widely distributed throughout the hepatic lobule, while HWS and HPI diets promoted reductions in liver steatosis. The effectiveness of HWS for modulating lipid metabolism was greater than that of HPI, as measured by plasma cholesterol reduction and liver steatosis.

What makes a species central in a cleaning mutualism network?

Mutualisms often form networks of interacting species, characterized by the existence of a central core of species that potentially drive the ecology and the evolution of the whole community. Centrality measures allow quantification of how central or peripheral a species is within a network, thus informing about the role of each species in network organization, dynamics, and stability. In the present study we addressed the question whether the structural position of species in the network (i.e. their topological importance) relates to their ecological traits. We studied interactions between cleaner and client reef fishes to identify central and peripheral species within a mutualistic network, and investigated five ecological correlates. We used three measures to estimate the level of centrality of a species for distinct structural patterns, such as the number of interactions and the structural proximity to other species. Through the use of a principal component analysis (PCA) we observed that the centrality measures were highly correlated (92.5%) in the studied network, which indicates that the same species plays a similar role for the different structural patterns. Three cleaner and ten client species had positive values of centrality, which suggests that these species are modulating ecological and evolutionary dynamics within the network. Higher centralities were related to higher abundances and feeding habits for client fishes, but not for cleaners. The high correlation between centrality measures in the present study is likely related to the nested structure of the cleaning network. The cleaner species` set, by having central species that are not necessarily the most abundant ones, bears potentially more vulnerable points for network cohesiveness. Additionally, the present study generalizes previous findings for plant-animal mutualisms, as it shows that the structure of marine mutualisms is also related to a complex interplay between abundance and niche-related features.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 30-untranslated region (3`-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3`-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety- like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety- related disorders in humans requires further investigation. Molecular Psychiatry (2009) 14, 894-905; doi: 10.1038/mp.2008.43; published online 22 April 2008

The relation between action, predictability and temporal contiguity in temporal binding

Previous studies have documented a subjective temporal attraction between actions and their effects. This finding, named intentional binding, is thought to be the result of a cognitive function that links actions to their consequences. Although several studies have tried to outline the necessary and sufficient conditions for intentional binding, a quantitative comparison between the roles of temporal contiguity, predictability and voluntary action and the evaluation of their interactions is difficult due to the high variability of the temporal binding measurements. In the present study, we used a novel methodology to investigate the properties of intentional binding. Subjects judged whether an auditory stimulus, which could either be triggered by a voluntary finger lift or be presented after a visual temporal marker unrelated to any action, was presented synchronously with a reference stimulus. In three experiments, the predictability, the interval between action and consequence and the presence of action itself were manipulated. The results indicate that (1) action is a necessary condition for temporal binding; (2) a fixed interval between the two events is not sufficient to cause the effect and (3) only in the presence of voluntary action do temporal predictability and contiguity play a significant role in modulating the effect.These findings are discussed in the context of the relationship between intentional binding and temporal expectation. (C) 2010 Elsevier B.V. All rights reserved.

Modulation of inflammatory response by selective inhibition of cyclooxygenase-1 and cyclooxygenase-2 in acute kidney injury

This work explored the role of inhibition of cyclooxygenases (COXs) in modulating the inflammatory response triggered by acute kidney injury. C57Bl/6 mice were used. Animals were treated or not with indomethacin (IMT) prior to injury (days -1 and 0). Animals were subjected to 45 min of renal pedicle occlusion and sacrificed at 24 h after reperfusion. Serum creatinine and blood urea nitrogen, reactive oxygen species (ROS), kidney myeloperoxidase (MPO) activity, and prostaglandin E2 (PGE(2)) levels were analyzed. Tumor necrosis factor (TNF)-alpha, t-bet, interleukin (IL)-10, IL-1 beta, heme oxygenase (HO)-1, and prostaglandin E synthase (PGES) messenger RNA (mRNA) were studied. Cytokines were quantified in serum. IMT-treated animals presented better renal function with less acute tubular necrosis and reduced ROS and MPO production. Moreover, the treatment was associated with lower expression of TNF-alpha, PGE(2), PGES, and t-bet and upregulation of HO-1 and IL-10. This profile was mirrored in serum, where inhibition of COXs significantly decreased interferon (IFN)-gamma, TNF-alpha, and IL-12 p70 and upregulated IL-10. COXs seem to play an important role in renal ischemia and reperfusion injury, involving the secretion of pro-inflammatory cytokines, activation of neutrophils, and ROS production. Inhibition of COX pathway is intrinsically involved with cytoprotection.

iNOS-derived Nitric Oxide Modulates Infection-stimulated Bone Loss

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS(-/-)). The iNOS(-/-) mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP(+)) osteoclasts were significantly more numerous in iNOS-/- mice. Furthermore, the increased bone resorption in iNOS(-/-) mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1 alpha (SDF-1 alpha/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-mu M verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and maybe considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases. (C) 2010 Elsevier Ltd and ISBI. All rights reserved.

Allogeneic apoptotic thymocyte-stimulated dendritic cells expand functional regulatory T cells

P>Dendritic cells (DCs) play an important role in the clearance of apoptotic cells. The removal of apoptotic cells leads to peripheral tolerance, although their role is still not clear. We show that the uptake of apoptotic thymocytes by DCs converts these cells into tolerogenic DCs resistant to maturation by lipopolysaccharide, modulating the production of interleukin-12 and up-regulating the expression of transforming growth factor-beta(1) latency associated peptide. We also observed that DCs pulsed with apoptotic cells in the allogeneic context were more efficient in the expansion of regulatory T cells (Tregs), and that this expansion requires contact between DCs and the T cell. The Tregs sorted from in vitro culture suppressed the proliferation of splenocytes in vitro in a specific and non-specific manner. In the in vivo model, the transfer of CD4+ CD25- cells to Nude mice induced autoimmunity, with cell infiltrate found in the stomach, colon, liver and kidneys. The co-transfer of CD4+ CD25- and CD4+ CD25+ prevented the presence of cell infiltrates in several organs and increased the total cell count in lymph nodes. Our data indicate that apoptotic cells have an important role in peripheral tolerance via induction of tolerogenic DCs and CD4+ CD25+ Foxp3+ cells that present regulatory functions.

5-HT(1B) receptor in the suprachiasmatic nucleus of the common marmoset (Callithrix jacchus)

Serotonin (5-HT) is involved in the fine adjustments at several brain centers including the core of the mammal circadian timing system (CTS) and the hypothalamic suprachiasmatic nucleus (SCN). The SCN receives massive serotonergic projections from the midbrain raphe nuclei, whose inputs are described in rats as ramifying at its ventral portion overlapping the retinohypothalamic and geniculohypothalamic fibers. In the SCN, the 5-HT actions are reported as being primarily mediated by the 5-HT1 type receptor with noted emphasis for 5-HT(1B) subtype, supposedly modulating the retinal input in a presynaptic way. In this study in a New World primate species, the common marmoset (Callithrix jacchus), we showed the 5-HT(1B) receptor distribution at the dorsal SCN concurrent with a distinctive location of 5-HT-immunoreactive fibers. This finding addresses to a new discussion on the regulation and synchronization of the circadian rhythms in recent primates. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Preventive and curative glycoside kaempferol treatments attenuate the TH2-driven allergic airway disease

Asthma is a chronic respiratory disease characterized by airway inflammation and airway hyperresponsiveness (AHR). One strategy to treat allergic diseases is the development of new drugs. Flavonoids are compounds derived from plants and are known to have antiallergic, anti-inflammatory, and antioxidant properties. To investigate whether the flavonoid kaempferol glycoside 3-O-[beta-D-glycopiranosil-(1 -> 6)-alpha-L-ramnopiranosil]-7-O-alpha-L-ramnopiranosil-kaempferol (GRRK) would be capable of modulating allergic airway disease (AAD) either as a preventive (GRRK P) or curative (GRRK C) treatment in an experimental model of asthma. At weekly intervals, BALB/c mice were subcutaneously (sc) sensitized twice with ovalbumin (OVA)/alum and challenged twice with OVA administered intranasally. To evaluate any preventive effects GRRK was administered 1 h (hour) before each OVA-sensitization and challenge, while to analyze the curative effects mice were first sensitized with OVA, followed by GRRK given at day 18 through 21. The onset: of AAD was evaluated 24 h after the last OVA challenge. Both treatments resulted in a dose-dependent reduction in total leukocyte and eosinophil counts in the bronchoalveolar lavage fluid (BAL). GRRK also decreased CD4(+), B220(+), MHC class II and CD40 molecule expressions in BAL cells. Histology and lung mechanic showed that GRRK suppressed mucus production and ameliorated the AHR induced by OVA challenge. Furthermore, GRRK impaired Th2 cytokine production (IL-5 and IL-13) and did not induce a Th1 pattern of inflammation. These findings demonstrate that GRRK treatment before or after established allergic lung disease down-regulates key asthmatic features. Therefore. GRRK has a potential clinical use for the treatment of allergic asthma. (C) 2009 Elsevier B.V. All rights reserved.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1 beta or TNF-alpha (tumour necrosis factor-a) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with P22(phox) antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.