PTGS2-related PGE2 affects oocyte MAPK phosphorylation and meiosis progression in cattle: late effects on early embryonic development

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.     

Overexpression of Brassica juncea wild-type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance

Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H(2) O(2) )-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis.     

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.     

Atypical/Nor98 scrapie infectivity in sheep peripheral tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrP(Sc) negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.     

PrP Expression Level and Sensitivity to Prion Infection

Mice overexpressing the prion protein (PrP) sequence from various host species are widely used for measuring infectious titers in prion disease. However, the impact that the transgene expression level might have on the susceptibility to infection raises some concerns about the final biological relevance of these models. Here we report that endpoint titration of a sheep scrapie isolate in sheep and in mice overexpressing the ovine PrP results in similar estimates of the infectious titer.     

Finding confidence limits on population growth rates: Bootstrap and analytic methods

When predicting population dynamics, the value of the prediction is not enough and should be accompanied by a confidence interval that integrates the whole chain of errors, from observations to predictions via the estimates of the parameters of the model. Matrix models are often used to predict the dynamics of age- or size-structured populations. Their parameters are vital rates. This study aims (1) at assessing the impact of the variability of observations on vital rates, and then on model’s predictions, and (2) at comparing three methods for computing confidence intervals for values predicted from the models. The first method is the bootstrap. The second method is analytic and approximates the standard error of predictions by their asymptotic variance as the sample size tends to infinity. The third method combines use of the bootstrap to estimate the standard errors of vital rates with the analytical method to then estimate the errors of predictions from the model. Computations are done for an Usher matrix models that predicts the asymptotic (as time goes to infinity) stock recovery rate for three timber species in French Guiana. Little difference is found between the hybrid and the analytic method. Their estimates of bias and standard error converge towards the bootstrap estimates when the error on vital rates becomes small enough, which corresponds in the present case to a number of observations greater than 5000 trees.     

Mutations in UDP-Glucose:Sterol Glucosyltransferase in Arabidopsis Cause Transparent Testa Phenotype and Suberization Defect in Seeds

In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis.Steryl glycosides (SGs) and acyl SGs (ASGs) are abundant constituents of the membranes of higher plants (Frasch and Grunwald, 1976; Warnecke and Heinz, 1994; Warnecke et al., 1997, 1999). SGs are synthesized by membrane-bound UDP-Glc:sterol glucosyltransferase (Hartmann-Bouillon and Benveniste, 1978; Ury et al., 1989; Warnecke et al., 1997), which catalyzes the glycosylation of the 3β-hydroxy group of sterols to produce a 3-β-d-glycoside. UGT80A2 has been found in the plasma membrane, Golgi vesicles, the endoplasmic reticulum membrane, and occasionally the tonoplast (Hartmann-Bouillon and Benveniste, 1978; Yoshida and Uemura, 1986; Ullmann et al., 1987; Warnecke et al., 1997). It has also been reported that a UDP-Glc-dependent glucosylceramide synthase from cotton (Gossypium hirsutum) is capable of synthesizing SG in plants (Hillig et al., 2003). All plant sterols can be glycosylated, given that sterol substrates are pathway end products (Δ⁵-sterols in Arabidopsis [Arabidopsis thaliana]) and not intermediates. The most commonly observed glycoside is Glc (Warnecke et al., 1997) but Xyl (Iribarren and Pomilio, 1985), Gal, and Man have been observed (Grunwald, 1978). Although rare in occurrence, SGs with di-, tri-, and tetraglucoside residues have also been reported (Kojima et al., 1989). SGs can be acylated, polyhydroxylated, or sulfated, but ASGs with fatty acids esterified to the primary alcohol group of the carbohydrate unit are the most common modifications (Lepage, 1964).SGs have been found as abundant membrane components in many species of plants, mosses, bacteria, fungi, and in some species of animals (Esders and Light, 1972; Mayberry and Smith, 1983; Murakami-Murofushi et al., 1987; Haque et al., 1996), yet relatively little is known about their biological functions. Because of the importance of sterols in membrane fluidity and permeability (Warnecke and Heinz, 1994; Warnecke et al., 1999; Schaller, 2003) and the phospholipid dependence of UDP-Glc:sterol glucosyltransferase (Bouvier-Nave et al., 1984), it has been postulated that SGs may have a role in adaptation to temperature stress (Palta et al., 1993). A difference in the proportion of glycosylated versus acylated sterols were reported in two different solanaceous species under the same cold acclimation experiment (Palta et al., 1993). In one species an increase in SG was correlated with a decrease in ASG. In contrast, the other species displayed no change in SG and ASG levels with cold acclimation conditioning. Hence, evidence for a role in temperature adaptation is lacking.Understanding the processes involved in SG production has additional human importance because SGs are highly bioactive food components and laboratory mice fed SGs faithfully lead to either amyotrophic lateral sclerosis or parkinsonism pathologies (Ly et al., 2007). Similarly, consumption of seeds of the cycad palm (Cycas micronesica), containing high SG levels, has been linked to an unusual human neurological disorder, amyotrophic lateral sclerosis-parkinsonism dementia complex, in studies of the people of Guam (Cruz-Aguado and Shaw, 2009). However, SG is a dominant moiety of all plant membranes and some of the most widely consumed plant products in the United States such as soybeans (Glycine max) have concentrations well within the dose range obtained by consumption of cycad seeds. Cholesterol glycoside is the SG most commonly identified in animal membranes and is exemplified by cases in snake epidermis cells (Abraham et al., 1987) and human fibroblast cells under heat shock (Kunimoto et al., 2000). Regarding a role for SGs in the membrane, in comparison to normal sterols, SG and ASG exchange more slowly between the monolayer halves of a bilayer, which could serve to regulate free sterol (FS) content and its distribution (Ullmann et al., 1987; Warnecke et al., 1999).A study of cellulose synthesis in herbicide-treated cotton fibers found that sitosterol β-glucoside (SSG) copurified with cellulose fragments (Peng et al., 2002), leading to speculation that SGs act as a primer for cellulose biosynthesis in higher plants (Peng et al., 2002). In support of the hypothesis, SSG biosynthesis was reported to be pharmacologically inhibited by the known cellulose biosynthesis inhibitor 2-6-dichlorobenzonitrile (DCB; Peng et al., 2002). However, in subsequent studies, DCB inhibition of cellulose synthesis was not reversed by the exogenous addition of SSG, and the effects of DCB on cellulose synthesis were so rapid that the turnover of SGs would need to be very fast to account for the effects of DCB on cellulose synthesis (DeBolt et al., 2007). Schrick et al. (2004) reported that sterol biosynthesis mutants fackel, hydra1, and sterol methyltransferase1/cephalopod have reduced levels of cellulose but a specific effect on SGs was not established. Hence, a role for SGs in plant growth and development remains speculative.Here we describe a genetic analysis of the biological roles of two isoforms of UDP-Glc:sterol glucosyltransferase, UGT80A2 and USGT80B1, that participate in the synthesis of SG in Arabidopsis. UDP-Glc-dependent glucosylceramide synthase may also be capable of synthesizing SG in plants (Hillig et al., 2003), but no analysis was performed herein. We show that mutations in one of these genes, UGT80B1, results in a lack of flavanoid accumulation in the seed coat and that it corresponds to transparent testa15 (tt15). Analysis of ugt80A2, ugt80B1, and a double mutant suggests that glycosylation of sterols by the UGT80A2 and UGT80B1 enzymes had no measurable consequence on cellulose levels in Arabidopsis seed, siliques, flowers, stems, trichomes, and leaves. Rather, we demonstrate that mutation of UGT80B1 principally alters embryonic development and seed suberin accumulation and cutin formation in the seed coat, leading to abnormal permeability and tetrazolium salt uptake.     

Modifications of plant cell activities by polypeptides secreted by Eutypa lata, a vineyard fungal pathogen

Eutypa dieback is a devastating disease induced in vineyards by the fungal pathogen Eutypa lata . The fungus colonizes the xylem tissues of trunk and cordons but is never found in the annual canes. Nevertheless, dwarfed shoots and leaf necrosis observed in diseased plants indicate that a necrotic signal can spread at a distance from the infected area. Eutypine, a small cyclic molecule, and related compounds have been postulated as the toxins inducing these symptoms. In this work, we evidence that E. lata secreted other metabolites of polypeptidic nature which induced toxic effects on canes and leaves of vines, and on leaves of other plant materials. The polypeptide fraction (PF) isolated from culture medium of mycelium induced transitory H⁺ fluxes and membrane depolarization of plant cells. Complementary assays with plasma membrane vesicles (PMV) showed that H⁺-ATPase is a primary site of action as indicated by inhibition of the enzyme activity and increase of H⁺ conductance of plasma membrane. The toxic effect was also obvious on respiration and photosynthesis. All these impairments led to a hindering in cell energetics and, as a consequence, to an inhibition of uptake of assimilates. Treatment with PF also triggered biological events, characteristics of elicitation as suggested by the early responses on cell membrane described above, the activation of NADPH oxidase and the activation of Phenylalanine ammonia lyase (PAL).     

Involvement of the Phospholipid Sterol Acyltransferase1 in Plant Sterol Homeostasis and Leaf Senescence

Genes encoding sterol ester-forming enzymes were recently identified in the Arabidopsis (Arabidopsis thaliana) genome. One belongs to a family of six members presenting homologies with the mammalian Lecithin Cholesterol Acyltransferases. The other one belongs to the superfamily of Membrane-Bound O-Acyltransferases. The physiological functions of these genes, Phospholipid Sterol Acyltransferase1 (PSAT1) and Acyl-CoA Sterol Acyltransferase1 (ASAT1), respectively, were investigated using Arabidopsis mutants. Sterol ester content decreased in leaves of all mutants and was strongly reduced in seeds from plants carrying a PSAT1-deficient mutation. The amount of sterol esters in flowers was very close to that of the wild type for all lines studied. This indicated further functional redundancy of sterol acylation in Arabidopsis. We performed feeding experiments in which we supplied sterol precursors to psat1-1, psat1-2, and asat1-1 mutants. This triggered the accumulation of sterol esters (stored in cytosolic lipid droplets) in the wild type and the asat1-1 lines but not in the psat1-1 and psat1-2 lines, indicating a major contribution of the PSAT1 in maintaining free sterol homeostasis in plant cell membranes. A clear biological effect associated with the lack of sterol ester formation in the psat1-1 and psat1-2 mutants was an early leaf senescence phenotype. Double mutants lacking PSAT1 and ASAT1 had identical phenotypes to psat1 mutants. The results presented here suggest that PSAT1 plays a role in lipid catabolism as part of the intracellular processes at play in the maintenance of leaf viability during developmental aging.Sterols are components of most eukaryotic membranes; as such, they are important regulators of membrane fluidity and thus influence membrane properties, functions, and structure (Demel and De Kruyff, 1976; Bloch, 1983; Schuler et al., 1991; Roche et al., 2008). Unlike animals, in which cholesterol is most often the unique end product of sterol biosynthesis, each plant species has its own distribution of sterols, with the three most common phytosterols being sitosterol, stigmasterol, and campesterol (Benveniste, 2004). In addition to their free sterol form, phytosterols are also found as conjugates, particularly fatty acyl sterol esters (SE). Since SE are hardly integrated into the bilayer of the membranes (Hamilton and Small, 1982), the biochemical process of sterol acylation is believed to play a crucial role in maintaining free sterol concentration in the cell membranes (Lewis et al., 1987; Dyas and Goad, 1993; Chang et al., 1997; Sturley, 1997; Schaller, 2004). In other words, SE are generally thought to constitute a storage pool of sterols when those are present in amounts greater than immediately required for the cells. For instance, in plants, accumulation of SE has been described during seed maturation and senescence or when plant cell cultures reach stationary phase (Dyas and Goad, 1993, and refs. therein) as well as in mutant lines overproducing sterols (Gondet et al., 1994; Schaller et al., 1995).In mammals and yeast, the genes involved in sterol esterification have been known for a long time. These genes encode two types of enzymes responsible for the formation of SE in animals, the Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT) and the Lecithin:Cholesterol Acyltransferase (LCAT). ACAT, which catalyzes an acyl-CoA-dependent acylation, is a membrane-bound enzyme acting inside the cells (Chang et al., 1997). LCAT, which is evolutionarily unrelated to ACAT, catalyzes transacylation of acyl groups from phospholipids to sterols. It is a soluble enzyme present in the blood stream, where it is an important regulator of circulating cholesterol (Jonas, 2000). The budding yeast Saccharomyces cerevisiae has two enzymes of the ACAT type for the synthesis of SE (Yang et al., 1996).In plants, genes encoding enzymes responsible for SE formation have long been unknown. Based on biochemical studies, it was suggested that phospholipids and/or neutral lipids could serve as acyl donors (Garcia and Mudd, 1978a, 1978b; Zimowski and Wojciechowski, 1981a, 1981b). The identification in the Arabidopsis (Arabidopsis thaliana) genome of two genes involved in sterol esterification was based on homology searches. First, the phospholipid:sterol acyltransferase gene AtPSAT1 (At1g04010) was found to display consistent identity with the mammalian LCAT and then was biochemically characterized by expression in Arabidopsis (Noiriel, 2004; Banas et al., 2005). The encoded protein was shown to be associated with microsomal membranes and to catalyze the transfer of unsaturated fatty acyl groups from position sn-2 of phosphatidylethanolamine (and phosphatidylcholine to a lesser extent) to sterols. The preferred acceptor molecules of PSAT1 were cholesterol, a minor biosynthetic end product in Arabidopsis, then campesterol and sitosterol, the two main end products. Sterol coincubation studies performed with this microsomal enzymatic assay showed that sterol precursors such as cycloartenol or obtusifoliol, which were poor substrates when incubated alone, were preferentially acylated in the presence of sitosterol, suggesting an activation of the enzyme by sitosterol (Banas et al., 2005). Another sterol acyltransferase gene, AtASAT1 (At3g51970), was identified in a survey of members of the Arabidopsis superfamily of membrane-bound O-acyltransferases with a yeast ACAT mutant functional complementation approach (Chen et al., 2007). AtASAT1 encodes a protein structurally related to the animal and yeast ACATs. This enzyme was shown to prefer saturated fatty acyl-CoAs as acyl donors and cycloartenol as the acyl acceptor. Overexpression of AtASAT1 in seeds of Arabidopsis resulted in a strong accumulation of cycloartenol fatty acyl esters accompanied by an increase of the whole SE content of these seeds and, in spite of a slight decrease of the free sterol pool, an increase of the total sterol content of the transgenic seeds by up to 60% compared with that of the wild type (Chen et al., 2007). We took advantage of the availability of Arabidopsis T-DNA insertion mutants of these two genes to investigate their respective physiological roles. Here, we report on the involvement of AtPSAT1 in leaf senescence, its major contribution to SE formation in leaves and seeds, and also its essential role in free sterol homeostasis in these organs.     

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs

AIM: Calibration of impedance measurement was performed vs the Association Fran?oise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs. METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems). Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%). The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis. The impedance signal was attributable to E. coli in 99% of cases. All cultures containing E. coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus. CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E. coli in live bivalve shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method. Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.