Gibberellin-responsive and -insensitive dwarfing alleles on wheat performance in contrasting tillage systems

Near-isogenic lines (NILs) of winter wheat varying for alleles for reduced height (Rht), gibberellin (GA) response and photoperiod insensitivity (Ppd-D1a) in cv. Mercia background (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c+Ppd-D1a, Rht-D1c, Rht12) and cv. Maris Widgeon (rht (tall), Rht-D1b, Rht-B1c) backgrounds were compared to investigate main effects and interactions with tillage (plough-based, minimum-, and zero-tillage) over two years. Both minimum- and zero- tillage were associated with reduced grain yields allied to reduced harvest index, biomass accumulation, interception of photosynthetically active radiation (PAR), and plant populations. Grain yields were optimized at mature crop heights of around 740mm because this provided the best compromise between harvest index which declined with height, and above ground biomass which increased with height. Improving biomass with height was due to improvements in both PAR interception and radiation-use efficiency. Optimum height for grain yield was unaffected by tillage system or GA-sensitivity. After accounting for effects of height, GA insensitivity was associated with increased grain yields due to increased grains per spike, which was more than enough to compensate for poorer plant establishment and lower mean grain weights compared to the GA-sensitive lines. Although better establishment was possible with GA-sensitive lines, there was no evidence that this effect interacted with tillage method. We find, therefore, little evidence to question the current adoption of wheats with reduced sensitivity to GA in the UK, even as tillage intensity lessens.

Dynamic hydrological modeling in drylands with TRMM based rainfall

This paper introduces and evaluates DryMOD, a dynamic water balance model of the key hydrological process in drylands that is based on free, public-domain datasets. The rainfall model of DryMOD makes optimal use of spatially disaggregated Tropical Rainfall Measuring Mission (TRMM) datasets to simulate hourly rainfall intensities at a spatial resolution of 1-km. Regional-scale applications of the model in seasonal catchments in Tunisia and Senegal characterize runoff and soil moisture distribution and dynamics in response to varying rainfall data inputs and soil properties. The results highlight the need for hourly-based rainfall simulation and for correcting TRMM 3B42 rainfall intensities for the fractional cover of rainfall (FCR). Without FCR correction and disaggregation to 1 km, TRMM 3B42 based rainfall intensities are too low to generate surface runoff and to induce substantial changes to soil moisture storage. The outcomes from the sensitivity analysis show that topsoil porosity is the most important soil property for simulation of runoff and soil moisture. Thus, we demonstrate the benefit of hydrological investigations at a scale, for which reliable information on soil profile characteristics exists and which is sufficiently fine to account for the heterogeneities of these. Where such information is available, application of DryMOD can assist in the spatial and temporal planning of water harvesting according to runoff-generating areas and the runoff ratio, as well as in the optimization of agricultural activities based on realistic representation of soil moisture conditions.

Comparative analysis of performance and stability among composite cross populations, variety mixtures and pure lines of winter wheat in organic and conventional cropping systems

This study investigated the effects of increased genetic diversity in winter wheat (Triticum aestivum L.), either from hybridization across genotypes or from physical mixing of lines, on grain yield, grain quality, and yield stability in different cropping environments. Sets of pure lines (no diversity), chosen for high yielding ability or high quality, were compared with line mixtures (intermediate level of diversity), and lines crossed with each other in composite cross populations (CCPn, high diversity). Additional populations containing male sterility genes (CCPms) to increase outcrossing rates were also tested. Grain yield, grain protein content, and protein yield were measured at four sites (two organically-managed and two conventionally-managed) over three years, using seed harvested locally in each preceding year. CCPn and mixtures out-yielded the mean of the parents by 2.4% and 3.6%, respectively. These yield differences were consistent across genetic backgrounds but partly inconsistent across cropping environments and years. Yield stability measured by environmental variance was higher in CCPn and CCPms than the mean of the parents. An index of yield reliability tended to be higher in CCPn, CCPms and mixtures than the mean of the parents. Lin and Binns’ superiority values of yield and protein yield were consistently and significantly lower (i.e. better) in the CCPs than in the mean of the parents, but not different between CCPs and mixtures. However, CCPs showed greater early ground cover and plant height than mixtures. When compared with the (locally non-predictable) best-yielding pure line, CCPs and mixtures exhibited lower mean yield and somewhat lower yield reliability but comparable superiority values. Thus, establishing CCPs from smaller sets of high-performing parent lines might optimize their yielding ability. On the whole, the results demonstrate that using increased within-crop genetic diversity can produce wheat crops with improved yield stability and good yield reliability across variable and unpredictable cropping environments.

Fibrin activates GPVI in human and mouse platelets

The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability.

Activation of glycoprotein VI (GPVI) and C-type lectin-like receptor-2 (CLEC-2) underlies platelet activation by diesel exhaust particles and other charged/hydrophobic ligands

Platelets are activated by a range of stimuli that share little or no resemblance in structure to each other or to recognized ligands, including diesel exhaust particles (DEP), small peptides [4N1-1, Champs (computed helical anti-membrane proteins), LSARLAF (Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides (fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation of human and mouse platelets through the glycoprotein VI (GPVI)-FcR γ-chain complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using platelets from mice deficient in either or both of these receptors. In addition, all of these ligands stimulate tyrosine phosphorylation in GPVI/CLEC-2-double-deficient platelets, indicating that they bind to additional surface receptors, although only in the case of dextran sulfate does this lead to activation. DEP, fucoidan and dextran sulfate, but not the other agonists, activate GPVI and CLEC-2 in transfected cell lines as shown using a sensitive reporter assay confirming a direct interaction with the two receptors. We conclude that this miscellaneous group of ligands bind to multiple proteins on the cell surface including GPVI and/or CLEC-2, inducing activation. These results have pathophysiological significance in a variety of conditions that involve exposure to activating charged/hydrophobic agents.

Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2)

Fucoidan, a sulfated polysaccharide from Fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylations of Syk and Phospholipase C-γ2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ-chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets.

Tityus discrepans scorpion venom activates platelets through GPVI and a novel Src-dependent signaling pathway

In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbβ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.

Molecular basis of platelet activation by an alphaIIbbeta3-CHAMPS peptide.

The present study demonstrates that the alphaIIb-CHAMPS peptide induces platelet activation through integrin alphaIIbbeta3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR gamma-chain and Syk. The use of the alphaIIb-CHAMPS peptide to study integrin alphaIIbbeta3 function is compromised by non-integrin-mediated effects.

Differential roles of the PKC novel isoforms, PKCδ and PKCε, in mouse and human platelets

Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. Methodology/Principal Findings In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

GPVI and CLEC-2

The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin, which are exposed following vessel damage. Initiation of platelet activation is through an immunoreceptor tyrosine-based activation motif (ITAM). C-type lectin receptor 2 (CLEC-2), following engagement by its endogenous ligand, podoplanin, also mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. This chapter compares the signaling pathways of both receptors and their role in hemostasis and thrombosis. Platelets are also increasingly implicated in processes beyond hemostasis and thrombosis. One such process is the efficient separation of the lymphatic and blood vasculatures, which is dependent on CLEC-2-mediated platelet activation.

Comparison of CLEC-2 and GPVI signaling in platelets: the role of adaptor proteins

GPVI activates platelets through an ITAM pathway by activation of Src and Syk kinases leading to activation of PLCy2. CLEC-2 has been shown to activate platelets using an ITAM-like sequence in its cytoplasmic tail that is also dependent on Src and Syk kinases, but shows a partial rather than an absolute dependence on adapter SLP-76 for activation of PLCy2. The aim of this thesis is to understand some of the key differences in these signalling pathways. GPVI is in complex with FcRwhich contains the ITAM sequence (Yxx(L/I)x6−12Yxx(L/I)). These two tyrosines provide a docking site for the tandem-SH2 domains of Syk. In this thesis I show that CLEC-2 signalling through Syk is mediated by phosphorylation of the CLEC-2 YxxL sequence, receptor dimerisation and cross-linking by the Syk SH2 domains. I also show that the differential requirement for SLP-76 is not mediated by Gads. Both signalling pathways also show partial dependency for LAT. I also show that a novel protein, G6f, is not able to substitute for LAT in this signalling pathway and also exclude the LAT-family proteins PAG, LIME, LAX and NTAL as potential LAT replacements in platelet activation by GPVI. These results extend our understanding of platelet activation by CLEC-2.

GPVI and CLEC-2 in hemostasis and vascular integrity

The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin through an immunoreceptor tyrosine-based activation motif (ITAM)-regulated signaling pathway. ITAMs are characterized by two YxxL sequences separated by 6-12 amino acids and are found associated with several classes of immunoglobulin (Ig) and C-type lectin receptors in hematopoietic cells, including Fc receptors. Cross-linking of the Ig GPVI leads to phosphorylation of two conserved tyrosines in the FcR gamma-chain ITAM by Src family tyrosine kinases, followed by binding and activation of the tandem SH2 domain-containing Syk tyrosine kinase and stimulation of a downstream signaling cascade that culminates in activation of phospholipase Cgamma2 (PLCgamma2). In contrast, the C-type lectin receptor CLEC-2 mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. CLEC-2 is a receptor for podoplanin, which is expressed at high levels in several tissues, including type 1 lung alveolar cells, lymphatic endothelial cells, kidney podocytes and some tumors, but is absent from vascular endothelial cells and platelets. In this article, we compare the mechanism of platelet activation by GPVI and CLEC-2 and consider their functional roles in hemostasis and other vascular processes, including maintenance of vascular integrity, angiogenesis and lymphogenesis.

Differences in muscle activity during hand dexterity tasks between women with arthritis and a healthy reference group

Background. Impaired hand function is common in patients with arthritis and it affects performance of daily activities; thus, hand exercises are recommended. There is little information on the extent to which the disease affects activation of the flexor and extensor muscles during these hand-dexterity tasks. The purpose of this study was to compare muscle activation during such tasks in subjects with arthritis and in a healthy reference group. Methods. Muscle activation was measured in m. extensor digitorium communis (EDC) and in m. flexor carpi radialis (FCR) with surface electromyography (EMG) in women with rheumatoid arthritis (RA, n = 20), hand osteoarthritis (HOA, n = 16) and in a healthy reference group (n = 20) during the performance of four daily activity tasks and four hand exercises. Maximal voluntary isometric contraction (MVIC) was measured to enable intermuscular comparisons, and muscle activation is presented as %MVIC. Results. The arthritis group used a higher %MVIC than the reference group in both FCR and EDC when cutting with a pair of scissors, pulling up a zipper and—for the EDC—also when writing with a pen and using a key (p < 0.02). The exercise “rolling dough with flat hands” required the lowest %MVIC and may be less effective in improving muscle strength. Conclusions. Women with arthritis tend to use higher levels of muscle activation in daily tasks than healthy women, and wrist extensors and flexors appear to be equally affected. It is important that hand training programs reflect real-life situations and focus also on extensor strength.

Effect of poultry viscera meal inclusion in broiler diets in different rearing periods on performance, carcass and parts yields

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)