Myocardial meta-[125l]iodobenzylguanidine uptake in awake genetically hypertensive rats at different ages: an autoradiographic study.

The purpose of this work was to evaluate changes in myocardial meta-[125I]iodobenzylguanidine ([125I]MIBG) uptake and distribution with age in awake spontaneously hypertensive rats (SHR) with respect to Wistar-Kyoto (WKY) rats. Rats were randomly divided into two groups, one for measuring myocardial [125I]MIBG uptake and distribution 4 h after its injection and the second for evaluating myocardial catecholamine concentrations. Mean arterial blood pressure, cardiac hypertrophy index (heart/body weight ratio), and heart rate were significantly higher with increasing age in SHR compared with matched WKY rats. Myocardial catecholamine concentrations and turnover did not differ between the two strains and were significantly decreased with increasing age. Myocardial [125I]MIBG uptake determined by gamma counting was similar in WKY rats and SHR and did not vary significantly with age when expressed as uptake density. However, in both strains of rats, [125I]MIBG uptake determined by autoradiography was significantly greater at the base of the heart than at the apex and midventricular levels, and the uptake values of young rats were significantly higher than those of older rats. In 21-week-old WKY rats and SHR, the highest [125I]MIBG uptake values were found in the right ventricle. Thus, quantitative autoradiography allowed detection of significant changes in myocardial [125I]MIBG uptake and showed its heterogeneous distribution in the rat heart.     

Dietary antioxidants as inhibitors of the heme-induced peroxidation of linoleic acid: mechanism of action and synergism

In this work, a quantitative kinetic model for investigating the heme-induced peroxidation of linoleic acid and its inhibition by two important dietary antioxidants, quercetin and alpha-tocopherol, is developed. The main conclusions of this work are: (1) The time dependence of the lipid hydroperoxide concentration is critically dependent on the rate constant for lipid hydroperoxide cleavage, initial fraction of lipid hydroperoxides among the pool of conjugated dienes, and rate of heme degradation. (2) The lipophilic antioxidant alpha-tocopherol acts as a chain-breaking antioxidant that quickly reduces 1-2 eq of lipid peroxyl radicals (inhibition of propagation), whereas the more hydrophilic antioxidant quercetin is only marginally chain-breaking but capable of reducing 4-5 eq of iron-oxo initiator (inhibition of initiation). (3) Based on comparisons between experimental peroxidation curves and simulated curves assuming additivity, it can be concluded that combinations of alpha-tocopherol and quercetin are generally synergistic. The kinetic analysis and HPLC titrations of the antioxidants both suggest that synergism mainly arises from a capacity of alpha-tocopherol to regenerate some quercetin oxidation products still endowed with a reducing activity.     

Anthropogenic Landscape in Southeastern Amazonia: Contemporary Impacts of Low-Intensity Harvesting and Dispersal of Brazil Nuts by the Kayapó Indigenous People

Brazil nut, the Bertholletia excelsa seed, is one of the most important non-timber forest products in the Amazon Forest and the livelihoods of thousands of traditional Amazonian families depend on its commercialization. B. excelsa has been frequently cited as an indicator of anthropogenic forests and there is strong evidence that past human management has significantly contributed to its present distribution across the Amazon, suggesting that low levels of harvesting may play a positive role in B. excelsa recruitment. Here, we evaluate the effects of Brazil nut harvesting by the Kayapó Indigenous people of southeastern Amazonia on seedling recruitment in 20 B. excelsa groves subjected to different harvesting intensities, and investigated if management by harvesters influences patterns of B. excelsa distribution. The number of years of low-intensity Brazil nut harvesting by the Kayapó over the past two decades was positively related to B. excelsa seedling density in groves. One of the mechanisms behind the higher seedling density in harvested sites seems to be seed dispersal by harvesters along trails. The Kayapó also intentionally plant B. excelsa seeds and seedlings across their territories. Our results show not only that low-intensity Brazil nut harvesting by the Kayapó people does not reduce recruitment of seedlings, but that harvesting and/or associated activities conducted by traditional harvesters may benefit B. excelsa beyond grove borders. Our study supports the hypothesis that B. excelsa dispersal throughout the Amazon was, at least in part, influenced by indigenous groups, and strongly suggests that current human management contributes to the maintenance and formation of B. excelsa groves. We suggest that changes in Brazil nut management practices by traditional people to prevent harvesting impacts may be unnecessary and even counterproductive in many areas, and should be carefully evaluated before implementation.     

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase'' Npr1'' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.     

Identification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E(2)). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE/MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. They were identified from the cytosolic fractions of cells treated for 24h with mitogenic concentrations of 1, 30 and 500 pM of 17β-estradiol. Five biomarkers were up-regulated proteins, namely HSP 74, EF2, FKBP4, EF1 and GDIB and one was a down-regulated protein, namely K2C8. Three of these proteins, EF2, FKBP4 and K2C8 are implicated in a network centered on the estrogen receptors ESR1 and ESR2 as well as on AKT1. After the discovery phase, three biomarkers were selected to test the presence of estrogens using selected reaction monitoring (SRM). They were monitored using SRM after incubation of MCF-7/BOS in the presence of E(2) for confirmation or selected xenoestrogens. Daidzein, coumestrol and enterolactone induced an up-regulation of EF2 and FKPB4 proteins, while tamoxifen and resveratrol induced a down-regulation. The exposure of all phytoestrogens induced the down-regulation of K2C8. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures.     

Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta

Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.     

VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.     

A Simple Genetic Architecture Underlies Morphological Variation in Dogs

Domestic dogs exhibit tremendous phenotypic diversity, including a greater variation in body size than any other terrestrial mammal. Here, we generate a high density map of canine genetic variation by genotyping 915 dogs from 80 domestic dog breeds, 83 wild canids, and 10 outbred African shelter dogs across 60,968 single-nucleotide polymorphisms (SNPs). Coupling this genomic resource with external measurements from breed standards and individuals as well as skeletal measurements from museum specimens, we identify 51 regions of the dog genome associated with phenotypic variation among breeds in 57 traits. The complex traits include average breed body size and external body dimensions and cranial, dental, and long bone shape and size with and without allometric scaling. In contrast to the results from association mapping of quantitative traits in humans and domesticated plants, we find that across dog breeds, a small number of quantitative trait loci (≤3) explain the majority of phenotypic variation for most of the traits we studied. In addition, many genomic regions show signatures of recent selection, with most of the highly differentiated regions being associated with breed-defining traits such as body size, coat characteristics, and ear floppiness. Our results demonstrate the efficacy of mapping multiple traits in the domestic dog using a database of genotyped individuals and highlight the important role human-directed selection has played in altering the genetic architecture of key traits in this important species.