Protein phosphatase 2A regulatory subunits affecting plant innate immunity,energy metabolism,and flowering time – joint functions among B'η subfamily members

Protein phosphatase 2A (PP2A) is a heterotrimeric complex comprising a catalytic, scaffolding, and regulatory subunit. The regulatory subunits are essential for substrate specificity and localization of the complex and are classified into B/B55, B'', and B” non-related families in higher plants. In Arabidopsis thaliana, the close paralogs B''η, B''θ, B''γ, and B''ζ were further classified into a subfamily of B'' called B''η. Here we present results that consolidate the evidence for a role of the B''η subfamily in regulation of innate immunity, energy metabolism and flowering time. Proliferation of the virulent Pseudomonas syringae in B''θ knockout mutant decreased in comparison with wild type plants. Additionally, B''θ knockout plants were delayed in flowering, and this phenotype was supported by high expression of FLC (FLOWERING LOCUS C). B''ζ knockout seedlings showed growth retardation on sucrose-free medium, indicating a role for B''ζ in energy metabolism. This work provides insight into functions of the B''η subfamily members, highlighting their regulation of shared physiological traits while localizing to distinct cellular compartments.     

Aggregation risk prediction for antibodies and its application to biotherapeutic development

Aggregation is a common problem affecting biopharmaceutical development that can have a significant effect on the quality of the product, as well as the safety to patients, particularly because of the increased risk of immune reactions. Here, we describe a new high-throughput screening algorithm developed to classify antibody molecules based on their propensity to aggregate. The tool, constructed and validated on experimental aggregation data for over 500 antibodies, is able to discern molecules with a high aggregation propensity as defined by experimental criteria relevant to bioprocessing and manufacturing of these molecules. Furthermore, we show how this tool can be combined with other computational approaches during early drug development to select molecules with reduced risk of aggregation and optimal developability properties.     

Seed weevils living on the edge: pressures and conflicts over body size in the endoparasitic Curculio larvae

Abstract 1. Body size in parasitic insects can be subjected to contrasting selective pressures, especially if they complete their development within a single host. On the one hand, a larger body size is associated with a higher fitness. On the other hand, the host offers a discrete amount of resources, thus constraining the evolution of a disproportionate body size. 2. The present study used the weevil Curculio elephas as a study model. Larvae develop within a single acorn, feeding on its cotyledons, and larval body size is strongly related to individual fitness. 3. The relationship between larval and acorn size was negatively exponential. Larval growth was constrained in small acorns, which did not provide enough food for the weevils to attain their potential size. Larval size increased and levelled off in acorns over a certain size (inflexion point), in which cotyledons were rarely depleted. When there were more than one larva per acorn, a larger acorn was necessary to avoid food depletion. 4. The results show that C. elephas larvae are sometimes endoparasitic, living on the edge of host holding capacity. If they were smaller they could avoid food depletion more easily, but the fitness benefits linked to a larger size have probably promoted body size increase. The strong negative effects of conspecific competition may have possibly influenced female strategy of laying a single egg per seed. 5. Being larger and fitter, but always within the limits of the available host sizes, may be one main evolutionary dilemma in endoparasites.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

A systematic comparative and structural analysis of protein phosphorylation sites based on the mtcPTM database

mtcPTM is an online repository of human and mouse phosphosites in which data are hierarchically organized to preserve biologically relevant experimental information, thus allowing straightforward comparisons of phosphorylation patterns found under different conditions. The database also contains the largest available collection of atomic models of phosphorylatable proteins. Detailed analysis of this structural dataset reveals that phosphorylation sites are found in a heterogeneous range of structural and sequence contexts. mtcPTM is available on the web .     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.