In Vivo Phosphorylation of Ser21 and Ser83 during Nutrient-induced Activation of the Yeast Protein Kinase A (PKA) Target Trehalase

The readdition of an essential nutrient to starved, fermenting cells of the yeast Saccharomyces cerevisiae triggers rapid activation of the protein kinase A (PKA) pathway. Trehalase is activated 5–10-fold within minutes and has been used as a convenient reporter for rapid activation of PKA in vivo. Although trehalase can be phosphorylated and activated by PKA in vitro, demonstration of phosphorylation during nutrient activation in vivo has been lacking. We now show, using phosphospecific antibodies, that glucose and nitrogen activation of trehalase in vivo is associated with phosphorylation of Ser²¹ and Ser⁸³. Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation despite reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast protein phosphatase 2A, suggesting that lower PKA activity causes reduced trehalase dephosphorylation. Hence, phosphorylation of trehalase in vivo is not sufficient for activation. Deletion of the inhibitor Dcs1 causes constitutive trehalase activation and phosphorylation. It also enhances binding of trehalase to the 14-3-3 proteins Bmh1 and Bmh2, suggesting that Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1 and Bmh2 eliminates both trehalase activation and phosphorylation. Our results reveal that trehalase activation in vivo is associated with phosphorylation of typical PKA sites and thus establish the enzyme as a reliable read-out for nutrient activation of PKA in vivo.     

‘Genetics is not the issue’: Insurers on genetics and life insurance

Valorization of knowledge has been defined as a major challenge in the context of genomics as an emerging strategic research field. Valorization is a Dutch science-policy concept for what is elsewhere called science impact or the third mission of universities. This article describes the institutionalization of valorization policy in the Dutch genomics research system as a specific manifestation of a changing social contract between science and society, which mainly targets economic value creation and the stimulation of entrepreneurship. A societal debate has emerged in which this focus on economic aspects has been strongly criticized as one-sided. In response, policy-makers are willing to adopt a broader definition of valorization. On the basis of an analysis of valorization policies and practices in Dutch medical genomics, this article draws attention to two myths in this valorization debate.     

Binding site of a dextran-specific homogeneous IgM: thermodynamic and spectroscopic mapping by dansylated oligosaccharides

The hapten binding properties of the homogeneous mouse IgM secreted by MOPC-104E were investigated. Hapten-association constants were determined either by equilibrium and displacement equilibrium dialysis or by fluorometric titrations of the protein with the fluorescent derivatives of the haptens. For the latter type of measurements, several oligosaccharides were derivatized to the corresponding dansylhydrazones. The synthesis, generally applicable to oligosaccharides with free reducing ends, is described. Analysis of the thermodynamic parameters for the binding of 18 haptens forms the basis for proposing a model of the binding site of MOPC-104E. This model is supported and refined by results of the measurements of linear and circular polarization of the fluorescence of the dansylated haptens. The binding site is proposed to consist of a cavity with about 12-A depth, complementary to a terminal nonreducing nigerosyl group. At the entrance to this cavity, a further subsite is identified forming interactions of lower specificity with an additional glucose unit.     

Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes

Somatic cells have been proposed to be limited in the number of cell divisions they can undergo. This is thought to be a mechanism by which stem cells retain their integrity preventing disease. However, we have recently discovered intestinal crypt stem cells that persist for the lifetime of a mouse, yet divide every day. We now demonstrate biochemically that primary isolated Lgr5+ve stem cells contain significant telomerase activity. Telomerase activity rapidly decreases in the undifferentiated progeny of these stem cells and is entirely lost in differentiated villus cells. Conversely, asymmetric segregation of chromosomes has been proposed as a mechanism for stem cells to protect their genomes against damage. We determined the average cell cycle length of Lgr5+ve stem cells at 21.5 h and find that Lgr5+ve intestinal stem cells randomly segregate newly synthesized DNA strands, opposing the 'immortal strand' hypothesis.     

Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.     

Base pairing of anhydrohexitol nucleosides with 2,6-diaminopurine, 5-methylcytosine and uracil asbase moiety

Hexitol nucleic acids (HNAs) with modified bases (5-methylcytosine, 2,6-diaminopurine or uracil) were synthesized. The introduction of the 5-methylcytosine base demonstrates that N -benzoylated 5-methylcytosyl-hexitol occurs as the imino tautomer. The base pairing systems (G:CMe, U:D, T:D and U:A) obey Watson-Crick rules. Substituting hT for hU, hCMefor hC and hD for hA generally leads to increased duplex stability. In a single case, replacement of hC by hCMedid not result in duplex stabilization. This sequence-specific effect could be explained by the geometry of the model duplex used for carrying out the thermal stability study. Generally, polypurine HNA sequences give more stable duplexes with their RNA complement than polypyrimidine HNA sequences. This observation supports the hypothesis that, besides changes in stacking pattern, the difference in conformational stress between purine and pyrimidine nucleosides may contribute to duplex stability. Introduction of hCMeand hD in HNA sequences further increases the potential of HNA to function as a steric blocking agent.     

Non-destructive determination of maize leaf and canopy chlorophyll content

The objective of this study was to develop a rapid non-destructive technique to estimate total chlorophyll (Chl) content in a maize canopy using Chl content in a single leaf. The approach was (1) to calibrate and validate a reflectance-based non-destructive technique to estimate leaf Chl in maize; (2) to quantify the relative contribution of each leaf Chl to the total Chl in the canopy; and (3) to establish a relationship between leaf Chl content and total Chl in a maize canopy. The Red Edge Chlorophyll Index CI(red edge)=(R(NIR)/R(red edge))-1 based on reflectances, R, in the red edge (720-730nm) and near infrared (770-800nm) was found to be an accurate measure of maize leaf Chl. It was able to predict leaf Chl ranging from 10 to 805mgChlm(-2) with root mean-square error less than 38mgChlm(-2). Relationships between Chl content in each maize leaf and total canopy Chl content were established and showed that Chl in the collar leaf before silking or ear leaves explained more than 80% and 87% of the variation in total Chl in a maize canopy, respectively. Thus, non-destructive measurements of both reflectance and area of a single leaf (either collar or ear) can be used to accurately estimate total Chl content in a maize canopy.     

Soluble Tumor Necrosis Factor Receptor 1 and 2 Predict Outcomes in Advanced Chronic Kidney Disease: A Prospective Cohort Study

Background

Soluble tumor necrosis factor receptors 1 (sTNFR1) and 2 (sTNFR2) have been associated to progression of renal failure, end stage renal disease and mortality in early stages of chronic kidney disease (CKD), mostly in the context of diabetic nephropathy. The predictive value of these markers in advanced stages of CKD irrespective of the specific causes of kidney disease has not yet been defined. In this study, the relationship between sTNFR1 and sTNFR2 and the risk for adverse cardiovascular events (CVE) and all-cause mortality was investigated in a population with CKD stage 4-5, not yet on dialysis, to minimize the confounding by renal function.

Patients and methods

In 131 patients, CKD stage 4-5, sTNFR1, sTNFR2 were analysed for their association to a composite endpoint of all-cause mortality or first non-fatal CVE by univariate and multivariate Cox proportional hazards models. In the multivariate models, age, gender, CRP, eGFR and significant comorbidities were included as covariates.

Results

During a median follow-up of 33 months, 40 events (30.5%) occurred of which 29 deaths (22.1%) and 11 (8.4%) first non-fatal CVE. In univariate analysis, the hazard ratios (HR) of sTNFR1 and sTNFR2 for negative outcome were 1.49 (95% confidence interval (CI): 1.28-1.75) and 1.13 (95% CI: 1.06-1.20) respectively. After adjustment for clinical covariables (age, CRP, diabetes and a history of cardiovascular disease) both sTNFRs remained independently associated to outcomes (HR: sTNFR1: 1.51, 95% CI: 1.30-1.77; sTNFR2: 1.13, 95% CI: 1.06-1.20). A subanalysis of the non-diabetic patients in the study population confirmed these findings, especially for sTNFR1.

Conclusion

sTNFR1 and sTNFR2 are independently associated to all-cause mortality or an increased risk for cardiovascular events in advanced CKD irrespective of the cause of kidney disease.     

Dual targeting of antioxidant and metabolic enzymes to the mitochondrion and the apicoplast of Toxoplasma gondii

Toxoplasma gondii is an aerobic protozoan parasite that possesses mitochondrial antioxidant enzymes to safely dispose of oxygen radicals generated by cellular respiration and metabolism. As with most Apicomplexans, it also harbors a chloroplast-like organelle, the apicoplast, which hosts various biosynthetic pathways and requires antioxidant protection. Most apicoplast-resident proteins are encoded in the nuclear genome and are targeted to the organelle via a bipartite N-terminal targeting sequence. We show here that two antioxidant enzymes-a superoxide dismutase (TgSOD2) and a thioredoxin-dependent peroxidase (TgTPX1/2)-and an aconitase are dually targeted to both the apicoplast and the mitochondrion of T. gondii. In the case of TgSOD2, our results indicate that a single gene product is bimodally targeted due to an inconspicuous variation within the putative signal peptide of the organellar protein, which significantly alters its subcellular localization. Dual organellar targeting of proteins might occur frequently in Apicomplexans to serve important biological functions such as antioxidant protection and carbon metabolism.     

A Cyclic GMP Signalling Module That Regulates Gliding Motility in a Malaria Parasite

The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3′, 5′-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase δ (PDEδ), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase β (GCβ), showing that in ookinetes GCβ is an important source for cGMP, and that PDEδ is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.