Adenylyl cyclase type-VIII activity is regulated by G(betagamma) subunits

The Ca2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether Gi/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by mu-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G(betagamma) subunits inhibit AC-VIII activity, while constitutively active alphai/o subunits do not. Different Gbeta isoforms varied in their efficacies, with Gbeta1gamma2 or Gbeta2gamma2 being more efficient than Gbeta3gamma2 and Gbeta4gamma2, while Gbeta5 (transfected with gamma2) had no effect. As for the Ggamma subunits, Gbeta1 inhibited AC-VIII activity in the presence of all gamma subunits tested except for gamma5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of Gbetagamma dimers, or to reduce Gbetagamma plasma membrane anchorage, markedly attenuated the mu-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that Gbetagamma (originating from agonist activation of these receptors) and probably not Galphai/o subunits are involved in the agonist inhibition of AC-VIII.     

Structural and EPR/ENDOR/ESEEM spectroscopic investigations of a vanadomolybdate Keggin-type polyoxometalate in organic solvent

An EPR spectrum of as synthesized [G.A. Tsigdinos, C.J. Hallada, Inorg. Chem. 7 (1968) 437-441], orange colored, H₅PV₂Mo₁₀O₄₀ polyoxometalate showed the presence of a reduced vanadium(IV) addenda atom. Surprisingly, further ³¹P ENDOR (electron-nuclear double resonance) measurements indicated the absence of a phosphorous heteroatom leading to the suggestion that H₅V^VV^IVMo₁₁O₄₀ exists as a previously unrecognized impurity in the typically synthesized H₅PV₂Mo₁₀O₄₀ compound. H_5/4PV^VO₄V^IV/VMo₁₁O₃₆ was then synthesized in low yield (0.8 mol%) by omitting the addition of phosphate in a typical H₅PV₂Mo₁₀O₄₀ preparation. The molecular formulation and structure was supported by X-ray crystallography, infrared and mass spectrometry. Further use of EPR/ENDOR/ESEEM (electron-spin echo envelope modulation) allowed the formulation of [V^VV^IVMo₁₁O₄₀]⁵⁻ as [V^VO₄V^IVMo₁₁O₃₆]⁵⁻. Accordingly, the polyoxometalate has a heteroatom core with 11 molybdenum addenda and one VO²⁺ moiety at the polyoxometalate surface. The redox potential and the catalytic activity of the new vanadomolybdate polyoxometalate compound were essentially identical to the often-studied H₅PV₂Mo₁₀O₄₀ polyoxometalate isomeric mixture.     

Differential growth identified in salamander larvae half‐sib cohorts: Survival strategy?

In this study we describe the growth of several different larval cohorts (i.e. half-siblings of the same mother born on the same day) of a rare, xeric-adapted salamander Salamandra s. infraimmaculata Martens, 1885, under constant density and food conditions from birth to metamorphosis. The larvae spend the critical first phase of their lives in water, mostly in temporary ponds. Age and weight at metamorphosis were highly affected by varying food conditions. We have identified six different growth modes that these larvae use, both fast growing and slow growing. Each larval cohort was found to use 2-4 different such growth modes regardless of their initial weight. Fast growing modes (I-III) will enable larvae to survive dry years, and metamorphose bigger. Slow growing modes (IV-VI), used by 8% of the larval population, will enable survival only in rainy years. These last growth modes effect differential temporal dispersal in wet years by delaying the emergence of postmetamorphs onto land. Distribution of growth modes in the larval population is affected by food but not by density conditions. Late-born, fast-growing larvae will have an advantage in dry years being able to metamorphose and disperse, whereas the slow-growing larvae will survive only in wet years.     

Comparative study with two different enrichments in the culture media used in the disinfectant efficacy assay

Recent changes in Brazilian legislation for commercial disinfectants have been published due to the recent epidemic of nosocomial infections caused by rapidly growing mycobacteria (RGM) in many states of Brazil over the last 8 years. One of these documents requires that all the manufacturers provide evidence of efficacy of sterilizing and disinfectant products, used for semi critical medical devices, against the Mycobacterium bovis BCG Moreau and Mycobacterium abscessus subsp. bolletii INCQS 00594 strains by using the Confirmative in vitro Test for Determining Tuberculocidal Activity of Disinfectants recommended by the Association of Official Analytical Chemists. These changes have caused additional costs and increased problems for importation of enrichment products at national laboratories where disinfectant efficacy assay service is performed. Middlebrook ADC Enrichment (ADC) is provided by a unique manufacturer and used in the official protocol. The aim of the present study was to evaluate an alternative in house low-cost enrichment composed of fetal bovine serum and glucose (FBSG) with ADC for performance of disinfectant efficacy assay against mycobacteria. After obtaining the growth curves for M. abscessus ATCC 19977, M. abscessus subsp. bolletii INCQS 00594, Mycobacterium chelonae ATCC 35752, and Mycobacterium fortuitum ATCC 6841 by using ADC enrichment and FBSG in Kirchners and 7H9 culture media. Through statistical analysis via the Kruskal-Wallis test on the evaluation of microorganism growth rate, it was observed that there was no inhibition of RGM growth by any of the enrichments used. These results suggest that low-cost enrichment FBSG may be used as a potential substitute of ADC for composition of media for mycobacterial growth, including in disinfectant tests.     

Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.     

Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis

Background

Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ).

Methodology/Principal Findings

The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cell-derived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS.

Conclusion/Significance

Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity.     

Rethinking the relationship between nestedness and beta diversity: a comment on Baselga (2010)

Baselga [Partitioning the turnover and nestedness components of beta diversity. Global Ecology and Biogeography, 19 , 134–143, 2010] proposed pairwise (β_nes) and multiple‐site (β_NES) beta‐diversity measures to account for the nestedness component of beta diversity. We used empirical, randomly created and idealized matrices to show that both measures are only partially related to nestedness and do not fit certain fundamental requirements for consideration as true nestedness‐resultant dissimilarity measures. Both β_nes and β_NES are influenced by matrix size and fill, and increase or decrease even when nestedness remains constant. Additionally, we demonstrate that β_NES can yield high values even for matrices with no nestedness. We conclude that β_nes and β_NES are not true measures of the nestedness‐resultant dissimilarity between sites. Actually, they quantify how differences in species richness that are not due to species replacement contribute to patterns of beta diversity. Finally, because nestedness is a special case of dissimilarity in species composition due to ordered species loss (or gain), the extent to which differences in species composition is due to nestedness can be measured through an index of nestedness.