Prevalent role of Akt and ERK activation in cardioprotective effect of Ca2+ channel- and beta-adrenergic receptor blockers

We studied cardioprotective as well as Akt and extracellular signal-activated kinase (ERK) activating effect of a Ca(2+) antagonist and a beta-adrenergic receptor blocker during ischemia-reperfusion, and compared these properties of the substances with that of a poly(ADP-ribose) polymerase (PARP) inhibitor used as a positive control throughout the experiments. Langendorff-perfused isolated rat hearts were subjected to 25 min global ischemia followed by 45 min reperfusion, and recovery of energy metabolism as well as functional cardiac parameters were monitored. Although to varying extents, all substances improved recovery of creatine phosphate, ATP, intracellular pH, and reutilization of inorganic phosphate. These favorable changes were accompanied by improved recovery of heart function parameters and reduced infarct size. In addition and again to varying extents, all studied substances decreased oxidative damage (lipid peroxidation and protein oxidation), and activated Akt, glycogen synthase kinase (GSK)-3beta, and ERK1/2. Correlation between cardioprotective and kinase activating effectivity of the compounds proved to be statistically significant. Physiological significance of these kinase activations was established by demonstrating that inhibition of Akt by LY294002 and ERK1/2 by PD98059 compromised the cardioprotective effect of all the substances studied. In conclusion, we demonstrated for the first time that activation of phosphatidylinositol-3-kinase (PI-3K)-Akt and ERK2 pathways significantly contributed to cardioprotective effects of a Ca(2+) antagonist and a beta-adrenergic receptor blocker. Furthermore, we found a strong correlation between cardioprotective and kinase-activating potencies of the substances studied (Verapamil, Metoprolol and two PARP inhibitors), which indicated the potentiality of these kinases as drug-targets in the therapy of ischemic heart disease.     

Ascaris lumbricoides pseudocoelomic body fluid induces a partially activated dendritic cell phenotype with Th2 promoting ability in vivo

Dendritic cells (DCs) matured with helminth-derived molecules that promote Th2 immune responses do not follow conventional definitions of DC maturation processes. While a number of models of DC maturation by Th2 stimuli are postulated, further studies are required if we are to clearly define DC maturation processes that lead to Th2 immune responses. In this study, we examine the interaction of Th2-inducing molecules from the parasitic helminth Ascaris lumbricoides with the maturation processes and function of DCs. Here we show that murine bone marrow-derived DCs are partially matured by A. lumbricoides pseudocoelomic body fluid (ABF) as characterised by the production of IL-6, IL-12p40 and macrophage inflammatory protein 2 (MIP-2) but no enhanced expression of cluster of differentiation (CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II was observed. Despite these phenotypic characteristics, ABF-stimulated DCs displayed the functional hallmarks of fully matured cells, enhancing DC phagocytosis and promoting Th2-type responses in skin-draining lymph node cells in vivo. ABF activated Th2-associated extracellular signal-regulated kinase-1 and nuclear factor-kB intracellular signalling pathways independently of toll-like receptor 4. Taken together, we believe this is the first paper to demonstrate A. lumbricoides murine DC-Th cell-driven responses shedding further light on DC maturation processes by helminth antigens.     

Involvement of adenosine A2A receptors in engulfment-dependent apoptotic cell suppression of inflammation

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.     

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115?kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115?kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.     

A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii: SUBSTRATE SELECTION ACHIEVED THROUGH MULTIMERIZATION*

Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers'' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states.     

Methods for Cryopreservation of Guinea Fowl Sperm

Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet) and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide). The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining). Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols (based on in vitro assessment of post-thaw semen quality) were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively). During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.     

Characterisation of the progression of azaserine-induced rat pancreatic adenocarcinoma by proliferative cell nuclear antigen,basement membrane laminin and trypsinogen immunohistochemistry

The progression of azaserine-induced rat pancreatic adenocarcinoma (AC) was characterised using quantitative and semiquantitative immunohistochemistry for proliferating cell nuclear antigen (PCNA), basement membrane laminin (BML) and trypsinogen (TG). Samples were taken 5-20 months after initiation. High PCNA-labelling indices (PCNA LIs) were measured 5 months after the induction of atypical acinar cell nodules (AACNs), which decreased later and stagnated until a further decline in the month 10 adenomas. Then a second premalignant proliferative wave was observed (month 13) within the adenoma stage. Later, in month 20 differentiated ACs PCNA LIs fell to the host tissue level but were found highest in the month 20 anaplastic ACs indicating a switch to malignant proliferation. Month 20 invasive ACs showed a number of separate proliferative foci. In early AACNs, BML decreased and remained low till the local maximum in the month 13 adenoma. Invasive ACs did not express BML. Month 5 AACN and differentiated AC were TG deficient but anaplastic AC regained its TG expression. However invasive AC was again TG negative. These results are discussed in combination with our previous data on progressional changes of autophagic capacity and microvessel densities.