bFGF and VEGF synergistically enhance endothelial cytoprotection via decay-accelerating factor induction

The complement-regulatoryprotein decay-accelerating factor (DAF) can be upregulated onendothelial cells (EC) by protein kinase C (PKC)-dependent and-independent pathways. We hypothesized that basic fibroblast growthfactor (bFGF) might induce EC DAF expression, providing acytoprotective mechanism for angiogenic neovessels againstcomplement-mediated injury. Incubation of umbilical vein, aortic, anddermal EC with bFGF or vascular endothelial growth factor (VEGF)significantly increased DAF expression. Growth factor-induced ECproliferation was inhibited by PKC antagonists. In contrast, althoughPKC antagonists inhibited VEGF-induced DAF expression, bFGF-induced DAFwas unaffected. Investigation of mitogen-activated kinase (MAPK)pathways also revealed differences, with bFGF-induced DAF dependent onp44/42 and p38 MAPK and VEGF requiring activation of p38 MAPK alone.Upregulation of DAF by bFGF was functionally relevant, reducing C3deposition on EC after complement activation by 60% and resulting inmarked reduction in complement-mediated EC lysis. bFGF and VEGF weresynergistic in terms of DAF expression, resulting in enhancedcytoprotection. These observations reveal parallel PKC-dependent and-independent pathways regulating complement activation duringangiogenesis. Further elucidation of these pathways may provideimportant insights into innate cytoprotective mechanisms in endothelium.

     

High rates of energy expenditure and water flux in free-ranging Point Reyes mountain beavers Aplodontia rufa phaea

We measured water flux and energy expenditure in free-ranging Point Reyes mountain beavers Aplodontia rufa phaea by using the doubly labeled water method. Previous laboratory investigations have suggested weak urinary concentrating ability, high rates of water flux, and low basal metabolic rates in this species. However, free-ranging measurements from hygric mammals are rare, and it is not known how these features interact in the environment. Rates of water flux (210+/-32 mL d(-1)) and field metabolic rates (1,488+/-486 kJ d(-1)) were 159% and 265%, respectively, of values predicted by allometric equations for similar-sized herbivores. Mountain beavers can likely meet their water needs through metabolic water production and preformed water in food and thus remain in water balance without access to free water. Arginine-vasopressin levels were strongly correlated with rates of water flux and plasma urea : creatinine ratios, suggesting an important role for this hormone in regulating urinary water loss in mountain beavers. High field metabolic rates may result from cool burrow temperatures that are well below lower critical temperatures measured in previous laboratory studies and suggest that thermoregulation costs may strongly influence field energetics and water flux in semifossorial mammals.     

From foraging to farming in the southern Levant: the development of Epipalaeolithic and Pre-pottery Neolithic plant management strategies

This paper reviews the archaeobotanical record of the transition from foraging to farming in the southern Levant. The concise presentation of the published botanical evidence follows a critical assessment of: (a) the nature of Epipalaeolithic plant management strategies, (b) the place of the southern Levant in the polycentric development of Near Eastern plant cultivation and domestication, and (c) region-specific pathways for the emergence of domesticated crop “packages”. Some inferences are drawn and suggestions are made concerning the potential contribution of archaeobotanical research to questions of broader archaeological significance about socio-economic change in the southern Levant during the Pre-pottery Neolithic.     

The Arrestin Fold: Variations on a Theme

Endocytosis of ligand-activated plasma membrane receptors has been shown to contribute to the regulation of their downstream signaling. β-arrestins interact with the phosphorylated tail of activated receptors and act as scaffolds for the recruitment of adaptor proteins and clathrin, that constitute the machinery used for receptor endocytosis. Visual- and β-arrestins have a two-lobe, immunoglobulin-like, β-strand sandwich structure. The recent resolution of the crystal structure of VPS26, one of the retromer subunits, unexpectedly evidences an arrestin fold in this protein, which is otherwise unrelated to arrestins. From a functional point of view, VPS26 is involved in the retrograde transport of the mannose 6-P receptor from the endosomes to the trans-Golgi network. In addition to the group of genuine arrestins and Vps26, mammalian cells harbor a vast repertoire of proteins that are related to arrestins on the basis of their PFAM Nter and Cter arrestin- domains, which are named Arrestin Domain- Containing proteins (ADCs). The biological role of ADC proteins is still poorly understood. The three subfamilies have been merged into an arrestin-related protein clan.This paper provides an overall analysis of arrestin clan proteins. The structures and functions of members of the subfamilies are reviewed in mammals and model organisms such as Drosophila, Caenorhabditis, Saccharomyces and Dictyostelium.Key Words: Arrestins, Vps26, GPCR, retromer, trafficking, endocytosis.     

Use of linear mixed models for genetic evaluation of gestation length and birth weight allowing for heavy-tailed residual effects

Background

The distribution of residual effects in linear mixed models in animal breeding applications is typically assumed normal, which makes inferences vulnerable to outlier observations. In order to mute the impact of outliers, one option is to fit models with residuals having a heavy-tailed distribution. Here, a Student''s-t model was considered for the distribution of the residuals with the degrees of freedom treated as unknown. Bayesian inference was used to investigate a bivariate Student''s-t (BSt) model using Markov chain Monte Carlo methods in a simulation study and analysing field data for gestation length and birth weight permitted to study the practical implications of fitting heavy-tailed distributions for residuals in linear mixed models.

Methods

In the simulation study, bivariate residuals were generated using Student''s-t distribution with 4 or 12 degrees of freedom, or a normal distribution. Sire models with bivariate Student''s-t or normal residuals were fitted to each simulated dataset using a hierarchical Bayesian approach. For the field data, consisting of gestation length and birth weight records on 7,883 Italian Piemontese cattle, a sire-maternal grandsire model including fixed effects of sex-age of dam and uncorrelated random herd-year-season effects were fitted using a hierarchical Bayesian approach. Residuals were defined to follow bivariate normal or Student''s-t distributions with unknown degrees of freedom.

Results

Posterior mean estimates of degrees of freedom parameters seemed to be accurate and unbiased in the simulation study. Estimates of sire and herd variances were similar, if not identical, across fitted models. In the field data, there was strong support based on predictive log-likelihood values for the Student''s-t error model. Most of the posterior density for degrees of freedom was below 4. Posterior means of direct and maternal heritabilities for birth weight were smaller in the Student''s-t model than those in the normal model. Re-rankings of sires were observed between heavy-tailed and normal models.

Conclusions

Reliable estimates of degrees of freedom were obtained in all simulated heavy-tailed and normal datasets. The predictive log-likelihood was able to distinguish the correct model among the models fitted to heavy-tailed datasets. There was no disadvantage of fitting a heavy-tailed model when the true model was normal. Predictive log-likelihood values indicated that heavy-tailed models with low degrees of freedom values fitted gestation length and birth weight data better than a model with normally distributed residuals.Heavy-tailed and normal models resulted in different estimates of direct and maternal heritabilities, and different sire rankings. Heavy-tailed models may be more appropriate for reliable estimation of genetic parameters from field data.     

FYVE-dependent endosomal targeting of an arrestin-related protein in amoeba

Background

Visual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins.

Methodology and Principal Findings

A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation.

Significance

This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.     

Identification of oscillatory genes in somitogenesis from functional genomic analysis of a human mesenchymal stem cell model

During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 h period consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 h, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3a(tm1Amc) mutants but not in Dll3(pu) mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro.     

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu‐detoxification and Cu delivery for cytochrome c oxidase (cbb₃‐Cox) assembly depend on two distinct Cu‐exporting P_1B‐type ATPases. The low‐affinity CopA is suggested to export excess Cu and the high‐affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb₃‐Cox biogenesis. In most organisms, CopA‐like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI‐like ATPases is unknown. Here we identified a CopZ‐like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox‐sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu‐sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb₃‐Cox activity is reduced, suggesting that CopZ not only supplies Cu to P_1B‐type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI‐CopZ protein complex in native R. capsulatus membranes.