Corticotropin-releasing hormone induces proliferation and TNF-alpha release in cultured rat microglia via MAP kinase signalling pathways

We have previously demonstrated that corticotropin-releasing hormone (CRH) receptor 1 (CRH-R1) is functionally expressed in rat microglia. In the present study, we show that CRH, acting on CRH-R1, promoted cell proliferation and tumour necrosis factor-alpha (TNF-alpha) release in cultured rat microglia. Exogenous CRH resulted in an increase in BrdU incorporation compared with control cells, which was observed in a range of concentrations of CRH between 10 and 500 nm, with a maximal response at 50 nm. The effect of CRH on BrdU incorporation was inhibited by a CRH antagonist astressin but not by a cAMP-dependent protein kinase inhibitor H89. Exposure of microglial cells to CRH resulted in a transient and rapid increase in TNF-alpha release in a dose-dependent manner. In the presence of astressin, the effects of CRH on TNF-alpha release were attenuated. CRH effects on TNF-alpha release were also inhibited by specific inhibitors of MEK, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) (PD98059) or p38 mitogen-activated protein kinase (SB203580), but not by H89. Furthermore, CRH induced rapid phosphorylation of ERK and p38 kinases. Astressin, PD98059, and SB230580 were able to inhibit CRH-induced kinase phosphorylation. These results suggest that CRH induces cell proliferation and TNF-alpha release in cultured microglia via MAP kinase signalling pathways, thereby providing insight into the interactions between CRH and inflammatory mediators.     

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.     

Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice

Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII-deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.     

Catecholamines and in vitro growth of pathogenic bacteria: enhancement of growth varies greatly among bacterial species

The purpose of this study was to examine the effects of catecholamines on in vitro growth of a range of bacterial species, including anaerobes. Bacteria tested included: Porphyromonas gingivalis, Bacteriodes fragilis, Shigella boydii, Shigella sonnie, Enterobacter Sp, and Salmonella choleraesuis. The results of the current study indicated that supplementation of bacterial cultures in minimal medium with norepinephrine or epinephrine did not result in increased growth of bacteria. Positive controls involving treatment of Escherichia coli with catecholamines did result in increased growth of that bacterial species. The results of the present study extend previous observations that showed differential capability of catecholamines to enhance bacterial growth in vitro.     

Calcineurin anchoring and cell signaling

The targeting of phosphatase PP2B or calcineurin toward certain substrates synchronizes a variety of physiological processes. This review emphasizes how the targeting of calcineurin through interaction with various anchoring proteins facilitates phosphatase regulation of T-cell activation, neuronal excitability and cardiac hypertrophy.     

Catecholase activity associated with copper-S100B

This study addresses the spectroscopic properties and reactivity associated with the copper-loaded form of S100B isolated from bovine brain. Copper(II)-S100B displays EPR features typical of a type II copper center and is shown here to exhibit catecholase activity, the two-electron oxidation of catechols. The steady-state kinetics associated with the oxidation of several catecholamines has been probed in order to further characterize this activity. The evidence provided indicates that the catecholase chemistry is copper initiated. Superoxide dismutase has no effect on the rates of catecholamine oxidation catalyzed by Cu-S100B, establishing that superoxide is not produced during this reaction, ruling out an autoxidative mechanism. Addition of catalase to the Cu-S100B reaction with catechols reduces the amount of oxygen consumed by 50%, demonstrating that peroxide is released during this reaction. The release of peroxide is mechanistically distinct from the type III dinuclear copper proteins, catechol oxidase and tyrosinase.     

Hierarchy of polymorphic variation and desensitization permutations relative to beta 1- and beta 2-adrenergic receptor signaling

Agonist-promoted desensitization of G-protein-coupled receptors results in partial uncoupling of receptor from cognate G-protein, a process that provides for rapid adaptation to the signaling environment. This property plays important roles in physiologic and pathologic processes as well as therapeutic efficacy. However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human beta(1)-adrenergic receptor (beta(1)AR) or a gain-of-function polymorphic variant (beta(1)AR-Arg(389)), and the beta(2)-adrenergic receptor (beta(2)AR) or a loss-of-function polymorphic receptor (beta(2)AR-Ile(164)). Adenylyl cyclase activities were determined with multiple permutations of the possible states of the receptor: genotype, basal, or agonist stimulated and with or without agonist pre-exposure. For the beta(1)AR, the enhanced function of the Arg(389) receptor underwent less agonist-promoted desensitization compared with its allelic counterpart. Indeed, the effect of polymorphic variation on absolute adenylyl cyclase activities was such that desensitized beta(1)AR-Arg(389) signaling was equivalent to non-desensitized wild-type beta(1)AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type beta(2)AR underwent less desensitization compared with beta(2)AR-Ile(164), thus the heterogeneity in absolute signaling was markedly broadened by this polymorphism. Inverse agonist function was not affected by polymorphisms of either subtype. A general model is proposed whereby up to 10 levels of signaling by G-protein-coupled receptors can be present based on the influences of desensitization and genetic variation on coupling.     

Ploidy race distributions since the Last Glacial Maximum in the North American desert shrub, Larrea tridentata

• 1 A classic biogeographic pattern is the alignment of diploid, tetraploid and hexaploid races of creosote bush (Larrea tridentata) across the Chihuahuan, Sonoran and Mohave Deserts of western North America. We used statistically robust differences in guard cell size of modern plants and fossil leaves from packrat middens to map current and past distributions of these ploidy races since the Last Glacial Maximum (LGM).
• 2 Glacial/early Holocene (26–10 ¹⁴C kyr bp or thousands of radiocarbon years before present) populations included diploids along the lower Rio Grande of west Texas, 650 km removed from sympatric diploids and tetraploids in the lower Colorado River Basin of south‐eastern California/south‐western Arizona. Diploids migrated slowly from lower Rio Grande refugia with expansion into the northern Chihuahuan Desert sites forestalled until after ~4.0 ¹⁴C kyr bp . Tetraploids expanded from the lower Colorado River Basin into the northern limits of the Sonoran Desert in central Arizona by 6.4 ¹⁴C kyr bp . Hexaploids appeared by 8.5 ¹⁴C kyr bp in the lower Colorado River Basin, reaching their northernmost limits (~37°N) in the Mohave Desert between 5.6 and 3.9 ¹⁴C kyr bp .
• 3 Modern diploid isolates may have resulted from both vicariant and dispersal events. In central Baja California and the lower Colorado River Basin, modern diploids probably originated from relict populations near glacial refugia. Founder events in the middle and late Holocene established diploid outposts on isolated limestone outcrops in areas of central and southern Arizona dominated by tetraploid populations.
• 4 Geographic alignment of the three ploidy races along the modern gradient of increasingly drier and hotter summers is clearly a postglacial phenomenon, but evolution of both higher ploidy races must have happened before the Holocene. The exact timing and mechanism of polyploidy evolution in creosote bush remains a matter of conjecture.

     

Enhanced branching morphogenesis in mammary glands of mice lacking cell surface beta1,4-galactosyltransferase

Development of the mammary gland is influenced both by the systemic hormonal environment and locally through cell-cell and cell-extracellular matrix (ECM) interactions. We have previously demonstrated aberrant mammary gland morphogenesis in transgenic mice with elevated levels of the long isoform of beta1,4-galactosyltransferase 1 (GalT), a proportion of which is targeted to the plasma membrane, where it plays a role in cell-ECM interactions. Here, we show that mammary glands of mice lacking the long GalT isoform exhibit a complementary phenotype. Cell-surface GalT activity was reduced by over 60%, but because the short GalT isoform is intact, total GalT activity was reduced only slightly relative to wild type. Mammary glands from long GalT-null mice were characterized by excess branching, and this phenotype was accompanied by altered expression of laminin chains. Laminin alpha1 and alpha3 were reduced 2.4- and 3.0-fold, respectively, while expression of laminin gamma2 was elevated 2.3-fold. The expression and cleavage of laminin gamma2 have been correlated with branching and cell migration, and Western blotting revealed an altered pattern in gamma2 cleavage products in long GalT-null mammary glands. We then examined the expression of metalloproteases that cleave laminins or that have been shown to play a role in mammary gland morphogenesis. Expression of MT1-MMP, a membrane-bound protease that can cleave laminin gamma2, was elevated 5.5-fold in the long GalT-nulls. MMP 7 was also elevated 5.1-fold. Our results suggest that expression of surface GalT is important for the proper regulation of matrix expression and deposition, which in turn regulates the proper branching morphogenesis of the mammary epithelial ductal system.