Myodegeneration in EDA-A2 transgenic mice is prevented by XEDAR deficiency

EDA-A1 and EDA-A2 are members of the tumor necrosis factor family of ligands. The products of alternative splicing of the ectodysplasin (EDA) gene, EDA-A1 and EDA-A2 differ by an insertion of two amino acids and bind to distinct receptors. The longer isoform, EDA-A1, binds to EDAR and plays an important role in sweat gland, hair, and tooth development; mutations in EDA, EDAR, or the downstream adaptor EDARADD cause hypohidrotic ectodermal dysplasia. EDA-A2 engages the receptor XEDAR, but its role in the whole organism is less clear. We have generated XEDAR-deficient mice by gene targeting and transgenic mice expressing secreted forms of EDA-A1 or EDA-A2 downstream of the skeletal muscle-specific myosin light-chain 2 or skin-specific keratin 5 promoter. Mice lacking XEDAR were indistinguishable from their wild-type littermates, but EDA-A2 transgenic mice exhibited multifocal myodegeneration. This phenotype was not observed in the absence of XEDAR. Skeletal muscle in EDA-A1 transgenic mice was unaffected, but their sebaceous glands were hypertrophied and hyperplastic, consistent with a role for EDA-A1 in the development of these structures. These data indicate that XEDAR-transduced signals are dispensable for development of ectoderm-derived organs but might play a role in skeletal muscle homeostasis.     

Chromatographic separation of (E)- and (Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC–ESI-MS/MS

A selective, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of (E)-entacapone and (Z)-entacapone in human plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500 μL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50 mm × 4.6 mm, 5 μm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor (α) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for (E)- and (Z)-isomers respectively. The parent → product ion transitions for both the isomers (m/z 306.1 → 233.0) and IS (m/z 237.3 → 194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3–6076 ng/mL and 23.8–5960 ng/mL for (E)-entacapone and (Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for (E)- and (Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200 mg (E)-entacapone tablet formulation under fasting conditions.     

The role of bulk protein in local models of ion-binding to proteins: comparative study of KcsA, its semisynthetic analog with a locked-in binding site, and valinomycin

In studying ion-selectivity in biomaterials, it is common to study ion-protein interactions within a local neighborhood around the ion. This local system analysis for the S₂ site of KcsA, its semisynthetic analog, and valinomycin yields the free energy change in exchanging K⁺ with Na⁺ in quantitative agreement with the value obtained by considering ion-interactions with the entire system. But the energetics of ion binding in the local system and in the entire system differ significantly and lead to different conclusions regarding the physical basis of ion selectivity. For configurations sampled from an all-atom simulation, we show that the selectivity free energy can be decomposed into a contribution arising from interactions of the ion with its local neighborhood, ΔW_local, and a term arising from the field imposed on the ion and the binding site by the rest of the medium, ΔW_φ. The local contribution ΔW_local is numerically close to the actual free energy difference because the field contribution is small. The field contribution is small because of cancellation of inversely related ion-medium and site-medium interactions. Our analysis presents a rigorous foundation for the numerical success of the local system analysis and shows that its implications do not always hold for the entire protein.     

Intestinal helminths induce haematological changes in dogs from Jabalpur, India

The effect of canine intestinal helminths on the haematological profile of 200 dogs, of both sexes and variable age, visiting university veterinary clinics for routine examination was investigated. The dogs were assigned to parasitized (n?=?39) and non-parasitized (n?=?161) groups of animals. Coprological examination revealed a 19.5% prevalence of different species of the helminths. Of these animals, 10.25% had mixed infections with Ancylostoma caninum, Toxascaris spp. and Dipylidium caninum. The intensity of A. caninum infection was the highest, with mean egg counts of 951.43 (standard error 88.66), followed by Toxascaris 283.33 (standard error 116.81) and D. caninum. The parasitized animals had significantly lower levels of haemoglobin, packed cell volume and total erythrocyte counts than non-parasitized animals (P?相似文献   

Effects of sequential depositional basins on lake response to urban and agricultural pollution: a palaeoecological analysis of the Qu'Appelle Valley, Saskatchewan, Canada

1. Palaeolimnological analyses of fossil diatoms and pigments were conducted in four lakes of the Qu'Appelle Valley, Saskatchewan, Canada, to quantify the effect of upstream depositional basins on lake response to urban and agricultural human activities. Pasqua, Echo, Mission and Katepwa lakes exhibit similar modern limnological characteristics, lie sequentially downstream from urban point sources of growth‐limiting nitrogen (N), yet drain similarly large areas of farmland (38–40 × 10³ km²). 2. Analyses indicated that all lakes were naturally productive, contained eutrophic diatoms (i.e. Stephanodiscus niagarae, S. hantzchii, S. parvus and Aulacoseira granulata), and supported blooms of colonial (as myxoxanthophyll) and potentially toxic N‐fixing cyanobacteria (aphanizophyll), even prior to the onset of European settlement (ca. 1890) and urban development (ca. 1930). 3. The onset of agricultural practices ca. 1890 had only modest effects on algal communities in the Qu'Appelle lakes, with subtle increases in eutrophic diatom species (Pasqua, Mission and Katepwa lakes) and 25–50% increases in pigment‐inferred algal abundance (Echo, Mission and Katepwa lakes). 4. Despite naturally high production, total algal abundance (β‐carotene) in upstream Pasqua Lake increased by more than 350% after intense urbanization beginning ca. 1930, while eutrophic diatoms became more common and cyanobacteria populations increased ten‐fold. Principal components analysis (PCA) explained 64% of diatom variance, and identified three eras corresponding to baseline, pre‐agricultural communities (1776–1890), an era of high production (ca. 1925–1960) and recent variable community composition following tertiary treatment of urban sewage (ca. 1977–1990). 5. Analyses of three downstream lakes demonstrated that urban impacts following 1930 remained evident in fossil profiles of β‐carotene and myxoxanthophyll, but that large blooms of N‐fixing cyanobacteria were restricted to the past 25 years at downstream Mission and Katepwa lakes. Similarly, PCA showed that fossil diatom assemblages exhibited little directional variation until the 1970s. 6. Together, these analyses support the hypothesis that upstream lakes were effective at reducing the impacts of point‐source urban nutrients on downstream lakes. In contrast, diffuse agricultural activities had only limited impacts on water quality and these were less well ameliorated by upstream basins.     

Modulation of K11-linkage formation by variable loop residues within UbcH5A

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.     

Fifteen compelling open questions in plant cell biology

As scientists, we are at least as excited about the open questions—the things we do not know—as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such “rules” conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

We asked 15 experts to address what they consider to be the most compelling open questions in plant cell biology and these are their questions.