Anthropogenic effects on population genetics of phytophagous insects associated with domesticated plants

The hypothesis of isolation by distance (IBD) predicts that genetic differentiation between populations increases with geographic distance. However, gene flow is governed by numerous factors and the correlation between genetic differentiation and geographic distance is never simply linear. In this study, we analyze the interaction between the effects of geographic distance and of wild or domesticated status of the host plant on genetic differentiation in the bean beetle Acanthoscelides obvelatus. Geographic distance explained most of the among-population genetic differentiation. However, IBD varied depending on the kind of population pairs for which the correlation between genetic differentiation and geographic distance was examined. Whereas pairs of beetle populations associated with wild beans showed significant IBD (P < 10(-4)), no IBD was found when pairs of beetle populations on domesticated beans were examined (P= 0.2992). This latter result can be explained by long-distance migrations of beetles on domesticated plants resulting from human exchanges of bean seeds. Beetle populations associated with wild beans were also significantly more likely than those on domesticated plants to contain rare alleles. However, at the population level, beetles on cultivated beans were similar in allelic richness to those on wild beans. This similarity in allelic richness combined with differences in other aspects of the genetic diversity (i.e., IBD, allelic diversity) is compatible with strongly contrasting effects of migration and drift. This novel indirect effect of human actions on gene flow of a serious pest of a domesticated plant has important implications for the spread of new adaptations such as resistance to pesticides.     

Chemical modulation of VLA integrin affinity in human breast cancer cells

The fact that disruption of integrin-extracellular matrix contacts leads to cell death, has converted cell adhesion into a potential target for the control of invasive cancer. In this work, we studied the functional consequences of the interference with the activity of the very late activation antigen (VLA) family of integrins in human breast cancer cell lines of distinct malignancy. The alpha2beta1-mediated adhesion reduced the entry of highly malignant, hormone-independent breast cancer cells into apoptosis. Adhesion of breast cancer cells through the VLA integrins alpha2beta1 and alpha5beta1 was significantly reduced by an apoptosis-inducing natural triterpenoid, dehydrothyrsiferol (DT), when studied on low amounts of extracellular matrix. This effect was dose-dependent, not related to cell toxicity and not shared with apoptosis-inducing standard chemotherapeutics, such as doxorubicin and taxol. The compound did not affect either the cell surface expression level of VLA integrins or cell distribution of vinculin and actin during cell spreading. In addition, neither phosphorylation of the focal adhesion kinase pp125FAK on Tyr397 nor the protein kinase B (Akt/PKB) on Ser473 was significantly altered by DT. The integrin activation level, assessed by binding of soluble collagen to the alpha2beta1 integrin, was reduced upon cell treatment with DT. Importantly, the TS2/16, an anti-beta1 activating monoclonal antibody was able to rescue DT-treated cells from apoptosis. Since the activation state of integrins is increasingly recognized as an essential factor in metastasis formation, findings presented herein reveal that the chemical regulation of integrin affinity may be a potential therapeutic strategy in cancer therapy.     

TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs

We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K(f)) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH(2)O PIP at both 35 degrees C and 40 degrees C. Ventilation with 35 cmH(2)O PIP increased K(f) by 2.2-fold at 35 degrees C and 3.3-fold at 40 degrees C compared with baseline, but K(f) increased significantly with time at 40 degrees C with 9 cmH(2)O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in K(f). In TRPV4(-/-) knockout mice, the high PIP ventilation protocol did not increase K(f) at either temperature. We have also found that lung distention caused Ca(2+) entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4(-/-) and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4(-/-) lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation.     

Identification of an N-acetylglucosamine transporter that mediates hyphal induction in Candida albicans

The sugar N-acetylglucosamine (GlcNAc) plays an important role in nutrient sensing and cellular regulation in a wide range of organisms from bacteria to humans. In the fungal pathogen Candida albicans, GlcNAc induces a morphological transition from budding to hyphal growth. Proteomic comparison of plasma membrane proteins from buds and from hyphae induced by GlcNAc identified a novel hyphal protein (Ngt1) with similarity to the major facilitator superfamily of transporters. An Ngt1-GFP fusion was detected in the plasma membrane after induction with GlcNAc, but not other related sugars. Ngt1-GFP was also induced by macrophage phagocytosis, suggesting a role for the GlcNAc response in signaling entry into phagolysosomes. NGT1 is needed for efficient GlcNAc uptake and for the ability to induce hyphae at low GlcNAc concentrations. High concentrations of GlcNAc could bypass the need for NGT1 to induce hyphae, indicating that elevated intracellular levels of GlcNAc induce hyphal formation. Expression of NGT1 in Saccharomyces cerevisiae promoted GlcNAc uptake, indicating that Ngt1 acts directly as a GlcNAc transporter. Transport mediated by Ngt1 was specific, as other sugars could not compete for the uptake of GlcNAc. Thus, Ngt1 represents the first eukaryotic GlcNAc transporter to be discovered. The presence of NGT1 homologues in the genome sequences of a wide range of eukaryotes from yeast to mammals suggests that they may also function in the cellular processes regulated by GlcNAc, including those that underlie important diseases such as cancer and diabetes.     

The effect of acute moderate hypoxia on accumulated oxygen deficit during intermittent exercise in nonacclimatized men

The objective of this study was to determine the effect of acute moderate hypoxia and rest duration on performance and on the accumulated oxygen deficit (AOD) in high-intensity intermittent efforts. After preliminary tests, 2 groups of nonacclimatized men (resident at 690 m above sea level) carried out 3 randomized protocols of effort (EXP1, EXP2, and EXP5) on 3 different days. These tests were performed at acute moderate altitude (2,320 m) by the hypoxia group (H) and in normoxia by the normoxia group (N). During EXP1 the subjects ran a maximum of five 400-m sprints (90% intensity) on a treadmill, with a pause between efforts of 1 minute. In EXP2 and EXP5 the same protocol was repeated, increasing the rest period between sprints to 2 and 5 minutes, respectively. Lactate accumulation and exhaled gases were measured during the tests. Accumulated oxygen deficit was calculated for each sprint. The total AOD (SigmaAOD) for each type of protocol was determined to be the sum of the corresponding accumulated deficits. The AODs were influenced by the length of rest period (p < 0.05) but not by H. The increase in recovery time between sprints increased the SigmaAOD (7,843 +/- 4,435 vs. 7,137 +/- 2,117 ml; 11,013 +/- 4,616 vs. 9,931 +/- 2,731 ml; 12,611 +/- 4,594 vs. 12,907 +/- 3,085 ml for H and N in EXP1, EXP2, and EXP5, respectively). The AOD increased in value when the same sprint was compared from EXP1 to EXP5 (p < 0.05). The results obtained show that exposure to acute moderate altitude does not affect the anaerobic pathway contribution in intermittent high-intensity exercises. Performance during this type of repeated effort is not altered during acute exposure to moderate altitude, which should be taken into account when an acclimatizing period is not possible.     

The Mechanoenzymatic Core of Dynamin-related Protein 1 Comprises the Minimal Machinery Required for Membrane Constriction

Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Dynamin-related protein 1 (Drp1), a large GTPase of the dynamin superfamily, is the main mediator of mitochondrial fission. Like prototypical dynamin, Drp1 is composed of a mechanochemical core consisting of the GTPase, middle, and GTPase effector domain regions. In place of the pleckstrin homology domain in dynamin, however, Drp1 contains an unstructured variable domain, whose function is not yet fully resolved. Here, using time-resolved EM and rigorous statistical analyses, we establish the ability of full-length Drp1 to constrict lipid bilayers through a GTP hydrolysis-dependent mechanism. We also show the variable domain limits premature Drp1 assembly in solution and promotes membrane curvature. Furthermore, the mechanochemical core of Drp1, absent of the variable domain, is sufficient to mediate GTP hydrolysis-dependent membrane constriction.     

Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability

NADH cytochrome b₅ reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC₅₀ = ∼275 μm), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC₅₀ = 10.81 μm) and ZINC39395747 (IC₅₀ = 9.14 μm), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development.     

The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca²⁺ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca²⁺ levels in response to nitrate treatments were blocked by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343. In addition, increase in cytoplasmic Ca²⁺ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca²⁺ channel blockers or phospholipase C inhibitors. These results indicate that Ca²⁺ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca²⁺ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.Plants are sessile organisms that evolved sophisticated sensing and response mechanisms to adapt to changing environmental conditions. Calcium, a ubiquitous second messenger in all eukaryotes, has been implicated in plant signaling pathways (Harper et al., 2004; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Multiple abiotic and biotic cues elicit specific and distinct spatiotemporal patterns of change in the concentration of cytosolic Ca²⁺ ([Ca2+]_cyt) in plants (Sanders et al., 2002; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Abscisic acid and heat shock treatments cause a rapid intracellular Ca²⁺ increase that is preceded by a transient increase in the level of inositol 1,4,5-trisphosphate (IP₃; Sanchez and Chua, 2001; Zheng et al., 2012). Ca²⁺ signatures are detected, decoded, and transmitted to downstream responses by a set of Ca²⁺ binding proteins that functions as Ca²⁺ sensors (White and Broadley, 2003; Dodd et al., 2010).Nitrate is the main source of N in agriculture and a potent signal that regulates the expression of hundreds of genes (Wang et al., 2004; Vidal and Gutiérrez, 2008; Ho and Tsay, 2010). Despite progress in identifying genome-wide responses, only a handful of molecular components involved in nitrate signaling has been identified. Several pieces of evidence indicate that NITRATE TRANSPORTER1.1 (NRT1.1)/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3 (AtNPF6.3) is a nitrate sensor in Arabidopsis (Ho et al., 2009; Gojon et al., 2011; Bouguyon et al., 2015). NRT1.1/AtNPF6.3 is required for normal expression of more than 100 genes in response to nitrate in Arabidopsis roots (Wang et al., 2009). Downstream of NRT1.1/AtNPF6.3, CALCINEURIN B-LIKE INTERACTING SER/THR-PROTEINE KINASE8 (CIPK8) is required for normal nitrate-induced expression of primary nitrate response genes, and the CIPK23 kinase is able to control the switch from low to high affinity of NRT1.1/AtNPF6.3 (Ho et al., 2009; Hu et al., 2009; Ho and Tsay, 2010; Castaings et al., 2011). CIPKs act in concert with CALCINEURIN B-LIKE proteins, plant-specific calcium binding proteins that activate CIPKs to phosphorylate downstream targets (Albrecht et al., 2001). Early experiments using maize (Zea mays) and barley (Hordeum vulgare) detached leaves showed that nitrate induction of two nitrate primary response genes was altered by pretreating leaves with the calcium chelator EGTA or the calcium channel blocker LaCl₃ (Sakakibara et al., 1997; Sueyoshi et al., 1999), suggesting an interplay between nitrate response and calcium-related signaling pathways. However, the role of calcium as a second messenger in the nitrate signaling pathway has not been directly addressed.We show that nitrate treatments cause a rapid increase of IP₃ and [Ca2+]_cyt levels and that blocking phospholipase C (PLC) activity inhibits both IP₃ and [Ca2+]_cyt increases after nitrate treatments. We provide evidence that NRT1.1/AtNPF6.3 is required for increasing both IP₃ and [Ca2+]_cyt in response to nitrate treatments. Altering [Ca2+]_cyt or blocking PLC activity hinders regulation of gene expression of nitrate-responsive genes. Our results indicate that Ca²⁺ is a second messenger in the nitrate signaling pathway of Arabidopsis.