Inbreeding and inbreeding depression in endangered red wolves (Canis rufus)

In natural populations, the expression and severity of inbreeding depression can vary widely across taxa. Describing processes that influence the extent of inbreeding and inbreeding depression aid in our understanding of the evolutionary history of mating systems such as cooperative breeding and nonrandom mate selection. Such findings also help shape wildlife conservation theory because inbreeding depression reduces the viability of small populations. We evaluated the extent of inbreeding and inbreeding depression in a small, re‐introduced population of red wolves (Canis rufus) in North Carolina. Since red wolves were first re‐introduced in 1987, pedigree inbreeding coefficients (f) increased considerably and almost every wild born wolf was inbred (average f = 0.154 and max f = 0.383). The large inbreeding coefficients were due to both background relatedness associated with few founders and numerous close relative matings. Inbreeding depression was most evident for adult body size and generally absent for direct fitness measures such as reproductive success and survival; no lethal equivalents (LE = 0.00) were detected in juvenile survival. The lack of strong inbreeding depression in direct measures of fitness could be due to a founder effect or because there were no outbred individuals for comparison. Our results highlight the variable expression of inbreeding depression across traits and the need to measure a number of different traits when evaluating inbreeding depression in a wild population.     

Overstimulation of Glutamate Signals Leads to Hippocampal Transcriptional Plasticity in Hamsters

It’s known that neurons in mammalian hibernators are more tolerant to hypoxia than those in non-hibernating species and as a consequence animals are capable of awakening from the arousal state without exhibiting cerebral damages. In addition, evidences have suggested that euthermic hamster neurons display protective adaptations against hypoxia, while those of rats are not capable, even though molecular mechanisms involved in similar neuroprotective strategies have not been yet fully studied. In the present work, overstimulation of glutamatergic receptors NMDA recognized as one of the major death-promoting element in hypoxia, accounted for altered network complexity consistent with a moderate reduction of hippocampal neuronal survival (p < 0.05) in hamsters. These alterations appeared to be featured concomitantly with altered glutamatergic signaling as indicated by significant down-regulation (p < 0.01) of NMDAergic (NR2A) and AMPAergic (GluR1, R2) receptor subtypes together with the metabotropic mGluR5 subtype. Diminished mRNA levels were also reported for NMDA receptor binding factors and namely PSD95 plus DREAM, which exert positive and negative regulatory properties, respectively, on receptor trafficking events. Conversely, involvement of glutamatergic signaling systems on neuronal excitotoxicity was strengthened by the co-activation of GABA_AR-mediated effects as indicated by toxic morphological effects being notably reduced along with up-regulated GluR1, GluR2, mGluR5, DREAM, and Homer1c scaffold proteins when muscimol was added. Overall, these results point to a neuroprotective role of the GABAergic system against excitotoxicity episodes via DREAM-dependent inhibition of NMDA receptor and activation of AMPA receptor plus mGluR5, respectively, thus proposing them as novel therapeutic targets against cerebral ischemic damages in humans.     

Deeply conserved susceptibility in a multi‐host,multi‐parasite system

Variation in susceptibility is ubiquitous in multi‐host, multi‐parasite assemblages, and can have profound implications for ecology and evolution in these systems. The extent to which susceptibility to parasites is phylogenetically conserved among hosts can be revealed by analysing diverse regional communities. We screened for haemosporidian parasites in 3983 birds representing 40 families and 523 species, spanning ~ 4500 m elevation in the tropical Andes. To quantify the influence of host phylogeny on infection status, we applied Bayesian phylogenetic multilevel models that included a suite of environmental, spatial, temporal, life history and ecological predictors. We found evidence of deeply conserved susceptibility across the avian tree; host phylogeny explained substantial variation in infection status, and results were robust to phylogenetic uncertainty. Our study suggests that susceptibility is governed, in part, by conserved, latent aspects of anti‐parasite defence. This demonstrates the importance of deep phylogeny for understanding present‐day ecological interactions.     

Evaluation of analogues of GalNAc as substrates for enzymes of the mammalian GalNAc salvage pathway

Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-Acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans, and glycoproteins; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc, which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogues of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, galactokinase 2 (GK2) and uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogues that can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway: a modification on C6 is not tolerated by GK2; AGX1 can use all products of GK2 although with various efficiencies; and all analogues transformed into UDP-GalNAc analogues except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogues that could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detection on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogues can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism, and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.     

Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis

Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.     

Actin bundling via LIM domains

The LIM domain is defined as a protein-protein interaction module involved in the regulation of diverse cellular processes including gene expression and cytoskeleton organization. We have recently shown that the tobacco WLIM1, a two LIM domain-containing protein, is able to bind to, stabilize and bundle actin filaments, suggesting that it participates to the regulation of actin cytoskeleton structure and dynamics. In the December issue of the Journal of Biological Chemistry we report a domain analysis that specifically ascribes the actin-related activities of WLIM1 to its two LIM domains. Results suggest that LIM domains function synergistically in the full-length protein to achieve optimal activities. Here we briefly summarize relevant data regarding the actin-related properties/functions of two LIM domain-containing proteins in plants and animals. In addition, we provide further evidence of cooperative effects between LIM domains by transiently expressing a chimeric multicopy WLIM1 protein in BY2 cells.Key words: Actin-binding proteins, actin-bundling, cysteine-rich proteins, cytoskeleton, LIM domainThe LIM domain is a ≈55 amino acid peptide domain that was first identified in 1990 as a common cystein-rich sequence found in the three homeodomain proteins LIN-11, Isl1 and MEC-3. It has since been found in a wide variety of eukaryotic proteins of diverse functions. Animals possess several families of LIM proteins, with members containing 1–5 LIM domains occasionally linked to other catalytic or protein-binding domains such as homeodomain, kinase and SH3 domains. In contrast, plants only possess two distinct sets of LIM proteins. One is plant-specific and has not been functionally characterized yet. The other one comprises proteins that exhibit the same overall structure as the animal cystein rich proteins (CRPs), i.e., two very similar LIM domains separated by a ≈50 amino acid-long interLIM domain and a relatively short and variable C-terminal domain (Fig. 1A). The mouse CRP2 protein was the first CRP reported to interact directly with actin filaments (AF) and to stabilize the latter.¹ Identical observations were subsequently described for the chicken CRP1 and tobacco WLIM1 proteins.^2,3 In addition, these two proteins were shown to arrange AF into cables both in vitro and in vivo and thus join the list of actin bundlers.Open in a separate windowFigure 1Domain maps for wild-type WLIM1 (A) and GFP-fused chimeric 3xWLIM1 (B). A. WLIM1 basically comprises a short N-terminal domain (Nt), two LIM domains (LIM1 and LIM2), an interLIM spacer (IL) and a C-terminal domain (Ct). B. 3xWLIM1 consists of three tandem WLIM1 copies. This chimeric protein has been fused in C-terminus to GFP and transiently expressed in tobacco BY2 cells.To identify the peptide domains of WLIM1 responsible for its actin-related properties/activities, we generated domain-deleted and single domain variants and submitted them to a series of in vivo and in vitro assays.⁴ Localization experiments established that both LIM domains are required to efficiently target the actin cytoskeleton in tobacco BY2 cells. High-speed (200,000 g) cosedimentation data confirmed that the actin-binding activity of WLIM1 relies on its LIM domains. Indeed, the deletion of either the first or the second LIM domain respectively resulted in a 5-fold and 10-fold decrease of the protein affinity for AF. Importantly, each single LIM domain was found able to interact with AF in an autonomous manner, although with a reduced affinity compared to the wild-type WLIM1. Low-speed (12,500 g) cosedimentation data and electron microscopy observations revealed that the actin bundling activity of WLIM1 is also triggered by its LIM domains. Surprisingly each single LIM domain was able to bundle AF in an autonomous manner, suggesting that WLIM1 has two discrete actin-bundling sites. However, the bundles induced by the variants containing only one LIM domain, i.e., LIM domain-deleted mutants and single LIM domains, differed from those induced by the full-length WLIM1. They appeared more wavy and loosely packed and formed only at relatively high protein:actin ratios. Together these data suggest that LIM domains are autonomous actin-binding and -bundling modules that function in synergy in wild-type WLIM1 to achieve optimal activities.To further assess the mechanism of cooperation between the LIM domains of plant CRP-related proteins, we generated a chimeric protein composed of three WLIM1 copies in tandem (3 × WLIM1, Fig. 1B), and transiently expressed it as a GFP-fusion in tobacco BY2 cells. We anticipated that such a six LIM domain-containing protein displays an even higher actin-bundling activity. (Fig. 2A) shows the typical actin cytoskeleton pattern in an expanding BY2 cell as visualized using the actin marker GFP-fABD2.⁵ As previously reported by Sheahan et al.,⁵ GFP-fABD2 decorated dense, transversely oriented, cortical networks as well as transvacuolar strands connecting the subcortical-perinuclear region to the cortex. Ectopic expression of WLIM1-GFP (BY2 cells normally do not express the WLIM1 gene) induced moderate but perceptible modifications of the actin cytoskeleton structure (Fig. 2B). Most AF are arranged in bundles thicker than those observed in GFP-fABD2 expressing cells and fine AF arrays are less frequently observed. As expected, this phenotype was significantly enhanced in cells transformed with the 3xWLIM1-GFP protein (Fig. 2C). Indeed, cells were almost devoided of fine AF arrays and exhibited very thick actin cables (Fig. 2C) that, at times (≈30 %), form atypical long looped structures (Fig. 2D). The appearance of such structures may result from the increase of cable stability and thickness induced by the 3xWLIM1-GFP protein, as these parameters are likely to determine, at least partially, the maximal length of actin bundles. Together the present observations support earlier data showing that LIM domains work in concert in LIM proteins to regulate actin bundling in plant cells. Strikingly, vertebrate and plant CRPs invariably contain two LIM domains. The lack, in these organisms, of CRP-related proteins combining more than two LIM domains may be explained by the fact that very thick cables, such as those induced by the artificial 3xWLIM1, may be too stable structures incompatible with the necessary high degree of actin cytoskeleton plasticity. As an exception, a muscle CRP-related protein with five LIM domains (Mlp84B) has been identified in Drosophila.⁶ However, rather than decorating actin filaments in an homogenous manner, this protein has been found to concentrate in a specialized region of the Z-discs where it stabilizes, in concert with D-titin, muscle sarcomeres.⁷Open in a separate windowFigure 2Typical actin cytoskeleton patterns in tobacco BY2 cells that have been transiently transformed, using a particle gun, with GFP-fABD2 (A), WLIM1-GFP (B), and 3xWLIM1-GFP (C and D). For each construct, more than 60 cells were analyzed by confocal microscopy. In the case of 3xWLIM1-GFP, two prevalent patterns have been observed (C and D). Bars = 20 µm.The relatively well conserved spacer length (≈50 amino acids) that separates the two LIM domains in vertebrate CRPs and related plant LIM proteins remains an intriguing feature the importance of which in actin cable organization remains to be established. Using electron microscopy we are currently evaluating the effects of the modification of the interLIM domain length on the structural properties of actin cables.