Natural cavity restoration as an alternative to nest box supplementation

Nest box supplementation is widely used to increase nest‐site availability for cavity nesting animals but the analysis of its effects on individuals breeding in natural cavities is often neglected. This study offers a novel restoration technique to revert abandonment of natural breeding sites by a secondary cavity avian bird, the European roller (Coracias garrulus), and other ecologically similar species. We found that, after a program of nest box supplementation with ensuing monitoring, rollers gradually abandon nesting in natural and seminatural cavities in favor of nest boxes because the latter are of higher quality. We examine whether reducing the entrance size of natural and seminatural cavities improves their suitability for rollers. A 6‐year program reduced the diameter of the entrance of sandstone cavities and cavities in bridges. This led to a high occupancy (59%) of manipulated nest‐sites. Manipulated sites were most frequently occupied by rollers and little owls (Athene noctua) (31 and 18% of sites, respectively). Manipulation did not affect clutch size or fledgling success. We suggest that nest‐site diversity and nesting in natural cavities should be preserved to reduce nest box dependence. Our study illustrates the value of nest boxes when used alongside restoration of natural breeding sites and provides insights for the management of natural cavities.     

Oxidation and nitrosylation of cysteines proximal to the intermediate filament (IF)-binding site of plectin: effects on structure and vimentin binding and involvement in IF collapse

As an intermediate filament (IF)-based cytolinker protein, plectin plays a key role in the maintenance of cellular cytoarchitecture and serves at the same time as a scaffolding platform for signaling cascades. Consisting of six structural repeats (R1-6) and harboring binding sites for different IF proteins and proteins involved in signaling, the plectin C-terminal domain is of strategic functional importance. Depending on the species, it contains at least 13 cysteines, 4 of which reside in the R5 domain. To investigate the structural and biological functions of R5 cysteines, we used cysteine-to-serine mutagenesis and spectroscopic, biochemical, and functional analyses. Urea-induced unfolding experiments indicated that wild-type R5 in the oxidized, disulfide bond-mediated conformation was more stable than its cysteine-free mutant derivative. The binding affinity of R5 for vimentin was significantly higher, however, when the protein was in the reduced, more relaxed conformation. Of the four R5 cysteines, one (Cys4) was particularly reactive as reflected by its ability to form disulfide bridges with R5 Cys1 and to serve as a target for nitrosylation in vitro. Using immortalized endothelial cell cultures from mice, we show that endogenous plectin is nitrosylated in vivo, and we found that NO donor-induced IF collapse proceeds dramatically faster in plectin-deficient compared with wild-type cells. Our data suggest an antagonistic role of plectin in nitrosylation (oxidative stress)-mediated alterations of IF cytoarchitecture and a possible role of R5 Cys4 as a regulatory switch.     

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.     

Bone-marrow mononuclear cell therapy of severe ischemic heart failure

We describe cell therapy for severe ischemic heart failure using transendocardial injection of autologous bone-marrow-derived mononuclear cells. The treated patients had significantly less heart failure and angina, sustained significant improvement of pumping power, exercise capacity, cardiac muscle irrigation, and blood supply to the body. Electrical and mechanical mappings of the myocardium before and after the therapy, and anatomopathological examination of the myocardium of one of the patients that had deceased of a stroke eleven months after the treatment indicated sustained neoangiogenesis and improvement of activity and quantity of cardiomyocytes in the injected regions. Post-hoc analyses of injected cell phenotype and improvement of myocardial function indicate that presence of CD8+ and CD56+ cells does not correlate with good prognostics, suggesting a possibility of cell selection. For 'no-option' severe cardiac patients, significant benefits of cell therapy and absence of adverse effects may justify the application of bone-marrow-derived cell therapy.     

Exogenous expression of caveolin-1 is sufficient for hepatic stellate cell activation

Caveolin-1 (Cav-1) expression is increased in hepatic stellate cells (HSC) upon liver cirrhosis and it functions as an integral membrane protein of lipid rafts and caveolae that regulates and integrates multiple signals as a platform. This study aimed to evaluate the role of Cav-1 in HSC. Thus, the effects of exogenous expression of Cav-1 in GRX cells, a model of activated HSC, were determined. Here, we demonstrated through evaluating well-known HSC activation markers – such as α-smooth muscle actin, collagen I, and glial fibrillary acidic protein – that up regulation of Cav-1 induced GRX to a more activated phenotype. GRX^EGFP-Cav1 presented an increased migration, an altered adhesion pattern, a reorganization f-actin cytoskeleton, an arrested cell cycle, a modified cellular ultrastructure, and a raised endocytic flux. Based on this, GRX ^EGFP-Cav1 represents a new cellular model that can be an important tool for understanding of events related to HSC activation. Furthermore, our results reinforce the role of Cav-1 as a molecular marker of HSC activation.     

Genome size and chromosomes in marine sponges [Suberites domuncula,Geodia cydonium]

The genome size of the marine sponges Suberites domuncula and Geodia cydonium has been determined by flow cytofluorometric analysis using diamidino-phenylindole [DAPI]. Using human lymphocytes as reference the amount of DNA in cells from S. domuncula has been determined to be 3.7 pg and that of G. cydonium 3.3 pg. While no chromosomes could be identified in G. cydonium, the karyotype of the Suberites domuncula is 32 chromsomes in the diploid state. The size of the chromosomes was between 0.25 and 1.0 μm. No pronounced banding pattern was visible.     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

Myelopoiesis in the omentum of normal mice and during abdominal inflammatory processes

Coelomic cavities are relatively isolated from the systemic circulation of blood cells. Resident cell populations have a proper phenotype and kinetics, maintaining their steady-state populations and their responsiveness to local inflammatory reactions, in which the number and quality of coelomic cells can be greatly increased and modified. We have addressed the question of whether the increase in cell infiltrate in the inflamed abdominal cavity is sustained by the proliferation of myeloid cells in the omentum, and if so what are the characteristics of the progenitor cells involved and how the omentum controls their proliferation and differentiation. In the omentum under normal conditions and with inflammation due to schistosomal infection we found that pluripotent early myeloid progenitors were capable of giving rise to all the myeloid lineages in clonogenic assays, but not to the totipotent blood stem cells. Besides the major haemopoietins (GM-CSF, M-CSF, G-CSF, IL-5), the omentum stroma constitutively expressed SDF-1 alpha, the chemokine which elicits homing of circulating early haemopoietic progenitors. While normal omentum stroma produced LIF, its expression was substituted by SCF in inflamed tissues. In the first situation a slow steady-state renewal of progenitors is potentially favoured, while their intense expansion may be predominant in the latter one. We propose that the increase in cells in the abdominal cavity in inflammatory reactions is due to the enhanced input and expansion of early myeloid progenitors sustaining the in situ production of abdominal cell populations, rather than to the input of systemic circulating inflammatory cells.     

Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin

Retinol is stored in liver, and the dynamic balance between its accumulation and mobilization is regulated by hepatic stellate cells (HSC). Representing less than 1% total liver protein, HSC can reach a very high intracellular retinoid (vitamin-A and its metabolites) concentration, which elicits their conversion from the myofibroblast to the fat-storing lipocyte phenotype. Circulating retinol is associated with plasma retinol-binding protein (RBP) or bovine serum albumin (BSA). Here we have used the in vitro model of GRX cells to compare incorporation and metabolism of BSA versus RBP associated [(3)H]retinol in HSC. We have found that lipocytes, but not myofibroblasts, expressed a high-affinity membrane receptor for RBP-retinol complex (KD = 4.93 nM), and both cell types expressed a low-affinity one (KD = 234 nM). The RBP-retinol complex, but not the BSA-delivered retinol, could be dislodged from membranes by treatments that specifically disturb protein-protein interactions (high RBP concentrations). Under both conditions, treatments that disturb the membrane lipid layer (detergent, cyclodextrin) released the membrane-bound retinol. RBP-delivered retinol was found in cytosol, microsomal fraction and, as retinyl esters, in lipid droplets, while albumin-delivered retinol was mainly associated with membranes. Disturbing the clathrin-mediated endocytosis did not interfere with retinol uptake. Retinol derived from the holo-RBP complex was differentially incorporated in lipocytes and preferentially reached esterification sites close to lipid droplets through a specific intracellular traffic route. This direct influx pathway facilitates the retinol uptake into HSC against the concentration gradients, and possibly protects cell membranes from undesirable and potentially noxious high retinol concentrations.