Functional dissection of XDppa2/4 structural domains in Xenopus development

The maintenance of pluripotency in mammalian embryonic stem cells depends upon the expression of regulatory genes like Oct3/4 and Sox2. While homologues of these genes are also characterized in non-mammalian vertebrates, like birds, amphibians and fish, existence and function of developmental pluripotency associated genes (Dppa) in lower vertebrates have not yet been reported. Here we describe a Dppa2/4-like gene, XDppa2/4, in Xenopus. The protein contains a SAP domain and a conserved C-terminal region. Overexpression of XDppa2/4, murine Dppa2 or Dppa4 produces similar phenotypes (defects in blastopore closure), while injection of XDppa2/4 morpholino generates a loss of blastopore closure and neural fold formation. Embryos die up to tailbud stage. mDppa2 (but not mDppa4) rescues blastopore closure and neurulation defects caused by XDppaMO, but does not prevent subsequent death of embryos. Although XDppa2/4 exhibits a Dppa-like expression pattern and is indispensable for embryogenesis, analyses of various marker genes make its role as a pluripotency factor rather unlikely. Both the gain and loss of function effects until the end of neurulation are caused by the conserved C-terminal region but not by the SAP domain. The SAP domain is required for association of XDppa2/4 to chromatin and for embryonic survival at later stages of development suggesting epigenetic programming events.     

mRuby,a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellular Structures

A monomeric variant of the red fluorescent protein eqFP611, mRuby, is described. With excitation and emission maxima at 558 nm and 605 nm, respectively, and a large Stokes shift of 47 nm, mRuby appears particularly useful for imaging applications. The protein shows an exceptional resistance to denaturation at pH extremes. Moreover, mRuby is about ten-fold brighter compared to EGFP when being targeted to the endoplasmic reticulum. The engineering process of eqFP611 revealed that the C-terminal tail of the protein acts as a natural peroxisomal targeting signal (PTS). Using an mRuby variant carrying the eqFP611-PTS, we discovered that ordered inheritance of peroxisomes is widespread during mitosis of different mammalian cell types. The ordered partitioning is realized by the formation of peroxisome clusters around the poles of the mitotic spindle and ensures that equal numbers of the organelle are inherited by the daughter cells. The unique spectral properties make mRuby the marker of choice for a multitude of cell biological applications. Moreover, the use of mRuby has allowed novel insights in the biology of organelles responsible for severe human diseases.     

MAPK signaling in H9c2 cardiomyoblasts exposed to cholesterol secoaldehyde – Role of hydrogen peroxide

3β-Hydroxy-5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known to be formed in ozone- and singlet oxygen-mediated oxidations of cholesterol, has been detected in the atherosclerotic plaque and in the brain of patients suffering from Alzheimer’s disease and Lewy body dementia. Previously, we have shown that, in H9c2 cardiomyoblasts, ChSeco induces oxidative stress followed by apoptosis involving both intrinsic and extrinsic signaling pathways. In the present study, we investigated the nature of reactive oxygen species (ROS) and its associated redox signaling in H9c2 cells upon treatment with ChSeco. Both catalase and deferoxamine, which lowered intracellular ROS, were found to alleviate the ChSeco-induced cytotoxicity. ChSeco-treated H9c2 cells showed a significant decrease in the intracellular catalase activity, suggesting the involvement of H₂O₂ in the associated cytotoxicity. Additionally, in ChSeco-exposed cells, there was a marked increase in lipid peroxidation and pre-treatment with SB 203580 (p38 MAPK inhibitor) and MEK1/2 inhibitor (ERK1/2 and JNK inhibitor) rendered protection against the cytotoxicity. An early increase in the expression of p-SAPK/JNK or delayed p38 MAPK did not alter ATF-2 but decreased c-Jun expression in these cells. Overall, these findings are consistent with MAPK signaling resulting from increased cellular H₂O₂ in ChSeco-induced cytotoxicity in cardiomyoblasts.     

Dynamin 3 is a component of the postsynapse,where it interacts with mGluR5 and Homer

The dynamins comprise a large family of mechanoenzymes known to participate in membrane modeling events. All three conventional dynamin genes (Dyn1, Dyn2, Dyn3) are expressed in mammalian brain and produce more than 27 different dynamin proteins as a result of alternative splicing. Past studies have suggested that Dyn1 participates in specialized neuronal functions such as rapid synaptic vesicle recycling, while Dyn2 may mediate the conventional clathrin-mediated uptake of surface receptors. Currently, the distribution, expression, and function of Dyn3 in neurons, or in any other cell type, are completely undefined. Here, we demonstrate that Dyn1 and Dyn3 localize differentially in the synapse. Dyn1 concentrates within the presynaptic compartment, while Dyn3 localizes to dendritic spine tips. Within the postsynaptic density (PSD), we found Dyn3, but not Dyn1, to be part of a biochemically isolated complex comprised of Homer and metabotropic glutamate receptors. Finally, although dominant-negative Dyn3 did not seem to inhibit receptor endocytosis, overexpression of a specific Dyn3 spliced variant in mature neurons caused a marked remodeling of dendritic spines. These data suggest that Dyn3 is a postsynaptic dynamin and, like its binding partner Homer, plays a significant role in dendritic spine morphogenesis and remodeling.     

Acoustic divergence between bottlenose dolphin whistles from the Central–Eastern North Atlantic and Mediterranean Sea

To improve our understanding of the complex genetic and ecological structure of bottlenose dolphin (Tursiops truncatus) populations, we examined the acoustic features of communication signals from two geographically contiguous areas: the Central–Eastern North Atlantic and the Mediterranean Sea. Variations in the whistles were evaluated for four locations. Ten signal parameters were measured and used to statistically differentiate between the areas. Over 79 % of sightings were correctly classified by discriminant function analysis, confirming an acoustic differentiation between the two basins. The results of cluster analysis using the mean values of the parameters for each sighting showed that the three easternmost sightings from the Mediterranean and one sighting from the Canary archipelago formed a separate cluster from the rest of the Atlantic. The two sightings from the Alboran Sea in the west Mediterranean were grouped with the Atlantic recordings. There was more variability in whistles from the Atlantic Ocean consistent with data from genetic and photo-identification studies that document resident and non-resident animals in the area. The results suggest that the Alboran area may be inhabited by animals differentiated from the rest of the Mediterranean basin as a result of habitat features.     

Energy allocation during concurrent pregnancy and lactation in Norway rats with delayed and undelayed implantation

Despite the high cost of lactation alone, concurrent pregnancy and lactation (CPL) is widespread among rodents. Many species that exhibit CPL delay implantation of the litter in utero while nursing. The first purpose of this study was to describe the pattern of energy allocation during CPL in a species with a large degree of overlap between gestation and lactation. Resting metabolic rate, food consumption and mass changes of Norway rat dams and litters, digestive efficiency and urinary energy loss of dams, and pup tissue energy equivalents were determined for CPL dams and for dams that were only lactating (C). CPL dams had significantly higher metabolic rates than C dams. Food consumption, pup growth, tissue energy equivalents, and assimilation efficiency were similar for both groups. The energy equivalent of mass change was greater for C dams, which gained in maternal mass (lipid) during lactation, than for CPL dams, which only increased in mass because of the litter in utero. The second purpose of this study was to investigate the suggestion that delayed implantation during CPL evolved as a mechanism to lower peak energy demands during CPL. Concurrently pregnant and lactating dams were injected with estrone (ECPL dams) on days 3-16 of lactation to prevent them from delaying implantation. A group of dams that were only lactating also received estrone injections (EC dams). ECPL dams produced smaller offspring at weaning than EC dams.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation with asymmetric gene flow during the nonsynchronous divergence of dry forest birds

Dry forest bird communities in South America are often fragmented by intervening mountains and rainforests, generating high local endemism. The historical assembly of dry forest communities likely results from dynamic processes linked to numerous population histories among codistributed species. Nevertheless, species may diversify in the same way through time if landscape and environmental features, or species ecologies, similarly structure populations. Here we tested whether six co‐distributed taxon pairs that occur in the dry forests of the Tumbes and Marañón Valley of northwestern South America show concordant patterns and modes of diversification. We employed a genome reduction technique, double‐digest restriction site‐associated DNA sequencing, and obtained 4407–7186 genomewide SNPs. We estimated demographic history in each taxon pair and inferred that all pairs had the same best‐fit demographic model: isolation with asymmetric gene flow from the Tumbes into the Marañón Valley, suggesting a common diversification mode. Overall, we also observed congruence in effective population size (N_e) patterns where ancestral N_e were 2.9–11.0× larger than present‐day Marañón Valley populations and 0.3–2.0× larger than Tumbesian populations. Present‐day Marañón Valley N_e was smaller than Tumbes. In contrast, we found simultaneous population isolation due to a single event to be unlikely as taxon pairs diverged over an extended period of time (0.1–2.9 Ma) with multiple nonoverlapping divergence periods. Our results show that even when populations of codistributed species asynchronously diverge, the mode of their differentiation can remain conserved over millions of years. Divergence by allopatric isolation due to barrier formation does not explain the mode of differentiation between these two bird assemblages; rather, migration of individuals occurred before and after geographic isolation.     

Putative roles of the CNF2 and CDTIII toxins in experimental infections with necrotoxigenic Escherichia coli type 2 (NTEC2) strains in calves

Newborn colostrum-restricted calves were orally inoculated with an Escherichia coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.