Alpha1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter

We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive (86)Rb(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine (30 microM) increased (86)Rb(+) influx. The NKCC inhibitor bumetanide (50 microM) reduced the response to phenylephrine by 45 +/- 13% (n = 12, P < 0.01). PD-98059 (10 microM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 +/- 13% (n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that alpha(1)-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after alpha(1)-AR stimulation was shown by immunoprecipitation of the phosphoprotein from (32)P(i) prelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by alpha(1)-AR stimulation through phosphorylation of this protein involving the ERK pathway.     

A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.     

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.     

SOCS-1 and SOCS-3 inhibit IFN-alpha-induced expression of the antiviral proteins 2,5-OAS and MxA

Although the use of IFN-alpha in combination with ribavirin has improved the treatment efficacy of chronic hepatitis C virus (HCV) infection, 20-50% of patients still fail to eradicate the virus depending on the HCV genotype. Recently, overexpression of HCV core protein has been shown to inhibit IFN signaling and induce SOCS-3 expression. Aim of this study was to examine the putative role of SOCS proteins in IFN resistance. By Western blot analysis, a 4-fold induction of STAT-1/3 phosphorylation by IFN-alpha was observed in mock-transfected HepG2 clones. In contrast, IFN-induced STAT-1/3 phosphorylation was considerably downregulated by SOCS-1/3 overexpression. In mock-transfected cells, IFN-alpha induced 2',5'-OAS and myxovirus resistance A (MxA) promoter activity 40- to 80-fold and 10- to 35-fold, respectively, and this effect was abrogated in SOCS-1/3 overexpressing cells. As detected by Northern blot technique, IFN-alpha potently induced 2',5'-OAS and MxA mRNA expression in the control clones. Overexpression of SOCS-1 completely abolished both 2',5'-OAS and MxA mRNA expression, whereas SOCS-3 mainly inhibited 2',5'-OAS mRNA expression. Our results demonstrate that SOCS-1 and SOCS-3 proteins inhibit IFN-alpha-induced activation of the Jak-STAT pathway and expression of the antiviral proteins 2',5'-OAS and MxA. These data suggest a potential role of SOCS proteins in IFN resistance during antiviral treatment.     

Sexual behavior and hormonal estrus cycles in captive aged lowland gorillas (Gorilla gorilla)

To evaluate whether observed cycles in proceptive behavior in aging lowland gorilla females (age 40+) at Brookfield Zoo were driven by ovarian activity, we compared monthly behavioral data to estradiol and progestogen cycles based on fecal hormone assessments. Progestogen peaks showed regularity and close coincidence with monthly sexual behaviors. Estradiol was more variable. Progestogen peaks varied between 22+/-5 days for the control female (29 years old), to 24+/-2.5 and 29+/-8 for the two aged subjects. In the first aged female, which was housed with other females and a silverback, the high degree of cyclicity in sexual behavior, regularity of progestogen cycles, and close concordance between hormonal cycling and sexual behavior strongly compared to patterns found (in this and other studies) in gorilla females <35 years old. Cyclical progestogen peaks were longer and more variable in the second aged female-perhaps because she lacked the social mediation of other females or a male. For husbandry reasons she is not housed with the gorilla group, behavioral data were not collected from her. The value of our longitudinal study is in obtaining reproductive profiles of primate females that are approaching maximum lifespan. This pilot study is part of a larger research project on reproductive senescence that will include other captive females >35 years old, a population that is rapidly increasing in North American zoos as gorillas continue to age.     

Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: I. Investigation of the UDP-GlcNAc 2-epimerase functionality

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design.     

A framework for modelling soil structure dynamics induced by biological activity

Soil degradation is a worsening global phenomenon driven by socio‐economic pressures, poor land management practices and climate change. A deterioration of soil structure at timescales ranging from seconds to centuries is implicated in most forms of soil degradation including the depletion of nutrients and organic matter, erosion and compaction. New soil–crop models that could account for soil structure dynamics at decadal to centennial timescales would provide insights into the relative importance of the various underlying physical (e.g. tillage, traffic compaction, swell/shrink and freeze/thaw) and biological (e.g. plant root growth, soil microbial and faunal activity) mechanisms, their impacts on soil hydrological processes and plant growth, as well as the relevant timescales of soil degradation and recovery. However, the development of such a model remains a challenge due to the enormous complexity of the interactions in the soil–plant system. In this paper, we focus on the impacts of biological processes on soil structure dynamics, especially the growth of plant roots and the activity of soil fauna and microorganisms. We first define what we mean by soil structure and then review current understanding of how these biological agents impact soil structure. We then develop a new framework for modelling soil structure dynamics, which is designed to be compatible with soil–crop models that operate at the soil profile scale and for long temporal scales (i.e. decades, centuries). We illustrate the modelling concept with a case study on the role of root growth and earthworm bioturbation in restoring the structure of a severely compacted soil.     

Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co‐regulate axonal outgrowth of mouse retinal ganglion cells

Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc‐dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad‐spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP‐deficient mice, disclosed that both MMP‐2 and MT1‐MMP, but not MMP‐9, are involved in this process. Furthermore, administration of a novel antibody to MT1‐MMP that selectively blocks pro‐MMP‐2 activation revealed a functional co‐involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP‐2 and MT1‐MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP‐2 and β1‐integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP‐2 and MT1‐MMP as promising axonal outgrowth‐promoting molecules.