Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.     

Mosquito immunity against Plasmodium

Understanding the molecular mechanisms of the innate immune responses of Anopheles gambiae against Plasmodium parasites is of great importance for current efforts to develop novel strategies for malaria disease control. The parasite undergoes substantial stage-specific losses during its development in the mosquito, which in some cases lead to complete refractoriness of the mosquito against the parasite. The underlying genetics of refractoriness are complex and multifactorial. Completion of the genome sequence of An. gambiae 2 years ago, together with the development of DNA microarrays in this species and the extension of the RNAi technique to adult mosquitoes, has allowed comparative and functional genomic approaches of the mosquito innate immune system. A variety of factors were shown to negatively affect the development of Plasmodium parasites in the mosquito, in some cases leading to complete transmission blockage. In addition, mosquito factors have been identified that play positive roles and are required for successful transmission of the parasite. These findings indicate a highly complex interplay between parasite and vector. Research is continuing to identify new factors involved in this interaction and to decipher the interplay of these molecules and their regulation.     

Expression of Rapeseed Microsomal Lysophosphatidic Acid Acyltransferase Isozymes Enhances Seed Oil Content in Arabidopsis

In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.With increasing environmental challenges and concerns, there is renewed interest in deriving plant-based sustainable alternatives for petroleum products, including carburants, lubricants, and industrial feed stocks. Modifying oilseed crops to produce oils of uniform composition containing fatty acids varying in chain length or possessing reactive functional groups is a primary objective (Jaworski and Cahoon, 2003), as is that of increasing the yield of seed oil (Lardizabal et al., 2008; Zheng et al., 2008). Early success in modifying seed oils to produce the more common fatty acids has been tempered by limited success in the production of high levels of unusual fatty acids (UFAs) in cultivated oilseeds (Thelen and Ohlrogge, 2002; Drexler et al., 2003). Such studies have led to the conclusion that in order to achieve levels of UFAs similar to those present in the oil of native species, enzymatic activities additional to fatty acid modification are necessary to optimize the synthesis (Mekhedov et al., 2001), stability (Eccleston and Ohlrogge, 1998), and channeling (Bafor et al., 1990) of the desired fatty acid into triacylglycerol (TAG).The synthesis of glycerolipids occurs in the cytoplasm using de novo-synthesized fatty acids exported from the plastid as acyl-CoA thioesters. The fatty acyl groups are incorporated into membrane and storage lipids by the sequential esterification of glycerol-3-phosphate by the action of glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) at sn-1 to form lysophosphatidic acid followed by lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) at sn-2 to form phosphatidic acid (PA; Somerville et al., 2000). Dephosphorylation of PA results in the formation of diacylglycerol (DAG), which in developing seeds may be directed into the production of TAG by acyl-CoA-independent reactions or by diacylglycerol acyltransferase (DAGAT; EC 2.3.1.20; Roscoe, 2005). The substrate preferences for acyl-thioesters and the selectivities for the acceptor molecules displayed by the microsomal acyltransferases play a crucial role in establishing the acyl composition of lipids (Frentzen, 1998). The TAG synthesized in most oilseeds of agronomic importance contains fatty acids that are the same as those present in cytoplasmic membrane lipids. In contrast, the seeds of species that synthesize TAGs with exotic fatty acid compositions possess microsomal acyltransferases that facilitate the incorporation of UFAs into storage lipids because of their broad GPAT and/or their selective DAGAT specificities (Wiberg et al., 1994; Frentzen, 1998). Furthermore, oilseeds characterized by TAGs that contain UFAs at sn-2 possess additional seed-specific microsomal LPAATs (Brown et al., 1995; Hanke et al., 1995; Knutzon et al., 1995) that exhibit a wide variation in substrate preference and that serve to ensure the channeling of UFAs to this position, thereby segregating incompatible fatty acids away from membrane lipids.Cloning of cDNAs from cultivated and exotic plants and the availability of entirely sequenced genomes from plant and algal species have revealed that a minimum of two classes of genes encoding microsomal LPAATs exist (Frentzen, 1998) within a larger, LPAAT-like gene family containing acyltransferases as yet functionally uncharacterized but distinct from GPATs (Roscoe, 2005). The class A microsomal LPAATs defined by Frentzen (1998) possess substrate preferences for C18:1-CoA typical of enzymes involved in membrane lipid synthesis and are ubiquitously expressed in the plant. In contrast, individual members of the class B LPAATs display preferences for distinct, unusual saturated or unsaturated acyl groups and are normally expressed in storage organs. Although class B LPAATs have been exploited to alter the stereochemical composition of rapeseed (Brassica napus) oil to permit the incorporation of modified fatty acids at sn-2 (Lassner et al., 1995; Knutzon et al., 1999), a significant increase in the total amount of UFAs was not accomplished by the expression of the class B LPAATs alone. In contrast, the transformation of rapeseed and Arabidopsis (Arabidopsis thaliana) with a yeast gene encoding a variant LPAAT, SLC1-1, capable of accepting very long chain fatty acyl (VLCFA)-CoA substrates resulted in an increase in the total VLCFAs and, unexpectedly, in total oil content (Zou et al. 1997).In our efforts to modify the fatty acid composition of oil in rapeseed, in particular to increase the content of VLCFAs, we have addressed the question of optimizing the environment for the correct functioning of LPAATs encoded by transgenes. The above studies using the various LPAAT transgenes indicate that channeling of UFAs into sn-2 of oilseed species remains problematic. The ability to obtain oils with uniform composition strongly depends on the occupancy of sn-2 by UFAs, yet the level of occupancy of sn-2 by fatty acids corresponding to the selectivity of the introduced LPAAT is variable and relatively modest. Occupancy of sn-2 is determined in part by the ability of the LPAAT encoded by the transgene to compete with the endogenous enzyme, a function of the acyl-CoA substrates available to the enzymes and the relative efficiencies of the enzymes to compete for the donor and acceptor substrates. We argued that there is latitude for the reduction of competing activities using an antisense strategy, and although microsomal LPAATs have been cloned from rapeseed, there are no reports of the characterization of the enzyme. Our objectives in this work were to identify and evaluate the potential contribution of LPAAT isozymes to TAG biosynthesis in rapeseed, thereby discerning targets for optimizing efforts to modify oils for industrial purposes. In this study, we catalogue a previously undescribed complexity in microsomal LPAAT diversity and identify a LPAAT isozyme likely to play an important role in TAG synthesis in rapeseed. In contrast to diverged LPAATs of plant origin, we demonstrate a positive effect of the overexpression of microsomal LPAATs on oil content and seed weight.     

Five labdane diterpenoids from the seeds of Aframomum zambesiacum

Five labdane diterpenoids, (3-5), zambesiacolactone A (7) and zambesiacolactone B (8), were isolated from the seeds of Aframomum zambesiacum (Baker) K. Schum., along with five known labdanes and a linear sesquiterpene, nerolidol. Their structures were elucidated by spectroscopic analysis. Their antiplasmodial activity was evaluated in vitro against Plasmodium falciparum. Compound 3 was the most active with an IC(50) value of 4.97 microM.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

The AutoDock suite at 30

The AutoDock suite provides a comprehensive toolset for computational ligand docking and drug design and development. The suite builds on 30 years of methods development, including empirical free energy force fields, docking engines, methods for site prediction, and interactive tools for visualization and analysis. Specialized tools are available for challenging systems, including covalent inhibitors, peptides, compounds with macrocycles, systems where ordered hydration plays a key role, and systems with substantial receptor flexibility. All methods in the AutoDock suite are freely available for use and reuse, which has engendered the continued growth of a diverse community of primary users and third‐party developers.     

Glucocorticoids increase osmotic water permeability (Pf) of neonatal rabbit renal brush border membrane vesicles

During postnatal maturation, there is an increase in renal brush border membrane vesicle (BBMV) osmotic water permeability and a parallel increase in aquaporin-1 (AQP1) protein abundance. The mechanisms responsible for these changes remain unknown. Because serum glucocorticoid levels rise postnatally and have previously been linked to other maturational changes in renal function, we examined the effects of glucocorticoids on osmotic (Pf) and diffusional (P(DW)) water permeability and AQP1 protein abundance of renal BBMV. Neonatal rabbits were treated with dexamethasone (10 microg/100 g) for three days and compared with control neonates and adults. Pf and P(DW) were measured at 20 degrees C with a stopped-flow apparatus using light-scattering and aminonaphthalene trisulfonic acid (ANTS) fluorescence, respectively. Pf was significantly higher in BBMV from dexamethasone-treated neonates compared with vehicle-treated neonates, but remained lower than in BBMV from adults (P<0.05). P(DW) in dexamethasone and vehicle-treated neonatal BBMV was lower than in adult BBMV. Pf/P(DW) ratio increased from neonate (5.1+/-0.3) to dexamethasone (7.0+/-0.1) and adult BBMV (6.3+/-0.1). AQP1 expression was increased by dexamethasone treatment to adult levels. Membrane fluidity, which is inversely related to generalized polarization (GP) of steady-state laurdan fluorescence, was significantly higher in neonatal BBMV than both dexamethasone and adult BBMV (GP: neonate 0.285+/-0.002, dexamethasone treatment 0.302+/-0.006, and adult 0.300+/-0.005; P<0.05). These combined results show that dexamethasone-treatment during days 4-7 of life increases BBMV water permeability despite a decrease in membrane fluidity. This occurs by increasing channel-mediated water transport, as reflected in an increase in AQP1 protein abundance and a higher Pf/P(DW) ratio. This mimics the maturational changes and suggests a physiological role for glucocorticoids in maturation of proximal tubule water transport.     

The clopidogrel after surgery for coronary artery disease (CASCADE) randomized controlled trial: clopidogrel and aspirin versus aspirin alone after coronary bypass surgery [NCT00228423]

Background

Saphenous vein graft disease remains a major limitation of coronary artery bypass graft surgery. The process of saphenous vein intimal hyperplasia begins just days after surgical revascularization, setting the stage for graft atherosclerotic disease and its sequalae. Clopidogrel improves outcomes in patients with atherosclerotic disease, and is effective at reducing intimal hyperplasia in animal models of thrombosis. Therefore, the goal of this study will be to evaluate the efficacy of clopidogrel and aspirin therapy versus aspirin alone in the prevention of saphenous vein graft intimal hyperplasia following coronary artery bypass surgery.

Methods

Patients undergoing multi-vessel coronary artery bypass grafting and in whom at least two saphenous vein grafts will be used are eligible for the study. Patients will be randomized to receive daily clopidogrel 75 mg or placebo, in addition to daily aspirin 162 mg, for a one year duration starting on the day of surgery (as soon as postoperative bleeding has been excluded). At the end of one year, all patients will undergo coronary angiography and intravascular ultrasound assessment of one saphenous vein graft as selected by randomization. The trial will be powered to test the hypothesis that clopidogrel and aspirin will reduce vein graft intimal hyperplasia by 20% compared to aspirin alone at one year following bypass surgery.

Discussion

This trial is the first prospective human study that will address the question of whether clopidogrel therapy improves outcomes and reduces saphenous vein graft intimal hyperplasia following cardiac surgery. Should the combination of clopidogrel and aspirin reduce the process of vein graft intimal hyperplasia, the results of this study will help redefine modern antiplatelet management of coronary artery bypass patients.     

Ionizing radiation induces a Yap1-dependent peroxide stress response in yeast

Repair of DNA damage is fundamental for cellular tolerance to ionizing radiation (IR) and many IR-induced DNA lesions are thought to occur as a result of oxidative stress. We investigated the physiological effects of IR in Saccharomyces cerevisiae by performing protein expression profiles in cells exposed to electron pulse irradiation. Transient induction of several antioxidant enzymes in wild-type cells, but not in cells lacking the oxidative stress regulator Yap1, indicated that IR exposure causes cellular oxidative stress. Yap1 activation involved oxidation to the intramolecular disulfide bond, a signature of activation by peroxide, and was dependent on the Yap1 peroxide sensor Orp1/Gpx3. H(2)O(2) was produced in the culture medium of irradiated cells and was both necessary and sufficient for IR-induced Yap1 activation. When IR was performed in the presence of N(2)O, obviating H(2)O(2) production and increasing hydroxyl radical ((*)OH) production, the Yap1 response was lost, indicating that Yap1 was unable to respond to (*)OH or (*)OH-induced damage. However, the Yap1 response to IR did not seem to be a primary determinant of cellular IR tolerance. Altogether, these data provide a molecular demonstration that cells experience in vivo peroxide stress during IR and indicate that the H(2)O(2) produced cannot account for IR toxicity.     

Low survival after release into the wild: assessing “the burden of captivity” on Mallard physiology and behaviour

Captive-reared animals used in reinforcement programs are generally less likely to survive than wild conspecifics. Digestion efficiency and naive behaviour are two likely reasons for this pattern. The Mallard is a species with high adaptability to its environment and in which massive reinforcement programs are carried out. We studied physiological and behavioural factors potentially affecting body condition and survival of captive-reared Mallards after being released. Digestive system morphology and an index of body condition were compared among three groups: captive-reared birds remaining in a farm (control), captive-reared birds released into the wild as juveniles (released) and wild-born birds (wild). We also compared behaviour and diet of released vs. wild Mallards. Finally, we conducted a 1-year survival analysis of captive-reared birds after release in a hunting-free area. Gizzard weight was lower in control Mallards, but the size of other organs did not differ between controls and wild birds. The difference in gizzard weight between released and wild birds disappeared after some time in the wild. Diet analyses suggest that released Mallards show a greater preference than wild for anthropogenic food (waste grain, bait). Despite similar time-budgets, released Mallards never attained the body condition of wild birds. As a consequence, survival probability in released Mallards was low, especially when food provisioning was stopped and during harsh winter periods. We argue that the low survival of released Mallards likely has a physiological rather than a behavioural (foraging) origin. In any case, extremely few released birds live long enough to potentially enter the breeding population, even without hunting. In the context of massive releases presently carried out for hunting purposes, our study indicates a low likelihood for genetic introgression by captive-reared birds into the wild population.