Oxidation of Se-Carboxymethyl-selenocysteine by L-aminoacid oxidase and by D-aspartate oxidase

Summary Se-Carboxymethyl-DL-selenocysteine (CMSeC) has been prepared in a pure crystalline form from selenocysteine and monochloroacetic acid. It has been shown that CMSeC is a substrate for the L-aminoacid oxidase from snake venom and for the D-aspartate oxidase from beef kidney. Oxygen consumption and ammonia production indicate that only the L or the D form of CMSeC are acted upon respectively by one or the other of the above enzymes. No noticeable differences were shown in the oxidation rate of CMSeC and S-carboxymethylcysteine, an indication that the substitution of a selenium for a sulfur atom in the molecule does not greatly affect the substrate specificity of the two enzymes. Data have been obtained suggesting that the product of the oxidative deamination of CMSeC is Se-carboxymethyl-selenopyruvic acid.     

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria

Summary We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained fromParamecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0m salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl^– than to K⁺ and is impermeable to Ca⁺⁺. The procedure for obtaining VDAC and the properties of the channel are highly reproducible.VDAC activity was found, upon fractionation of the paramecium membranes, to come from the mitochondria. We note that the published data on mitochondrial Cl^– permeability suggest that there may indeed be a voltage-dependent Cl^– permeability in mitochondria.The method of incorporating VDAC into planar lipid bilayers may be generally useful for reconstituting biological transport systems in these membranes.     

Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.     

Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis

A fast and reliable Multiplex-PCR assay was established to identify the species Lactobacillus johnsonii. Two opposing rRNA gene-targeted primers have been designed for this specific PCR detection. Specificity was verified with DNA samples isolated from different lactic acid bacteria. Out of 47 Lactobacillus strains isolated from different environments, 16 were identified as L. johnsonii by PCR. The same set of strains was investigated with five alternative molecular typing methods: enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR), amplified fragment length polymorphism, single triplicate arbitrarily primed PCR, and pulsed-field gel electrophoresis in order to compare the discriminatory power of these methods. The reported data strongly support the highly significant heterogeneity among all L. johnsonii isolates, potentially linked to their origin of isolation. The use of species-specific primers as well as rapid and highly powerful PCR-based molecular typing tools (namely ERIC- and REP-PCR techniques) should be respectively envisaged for identifying, differentiating and monitoring L. johnsonii strains from various environmental samples, for product monitoring, for species tracing in clinical studies as well as bacterial profiling of various microecological or gastrointestinal environments.     

Ocean acidification bends the mermaid's wineglass

Ocean acidification lowers the saturation state of calcium carbonate, decreasing net calcification and compromising the skeletons of organisms such as corals, molluscs and algae. These calcified structures can protect organisms from predation and improve access to light, nutrients and dispersive currents. While some species (such as urchins, corals and mussels) survive with decreased calcification, they can suffer from inferior mechanical performance. Here, we used cantilever beam theory to test the hypothesis that decreased calcification would impair the mechanical performance of the green alga Acetabularia acetabulum along a CO₂ gradient created by volcanic seeps off Vulcano, Italy. Calcification and mechanical properties declined as calcium carbonate saturation fell; algae at 2283 µatm CO₂ were 32% less calcified, 40% less stiff and 40% droopier. Moreover, calcification was not a linear proxy for mechanical performance; stem stiffness decreased exponentially with reduced calcification. Although calcifying organisms can tolerate high CO₂ conditions, even subtle changes in calcification can cause dramatic changes in skeletal performance, which may in turn affect key biotic and abiotic interactions.     

Are fire,soil fertility and toxicity,water availability,plant functional diversity,and litter decomposition related in a Neotropical savanna?

Understanding how biodiversity and ecosystem functioning respond to changes in the environment is fundamental to the maintenance of ecosystem function. In realistic scenarios, the biodiversity-ecosystem functioning path may account for only a small share of all factors determining ecosystem function. Here, we investigated the strength to which variations in environmental characteristics in a Neotropical savanna affected functional diversity and decomposition. We sought an integrative approach, testing a number of pairwise hypotheses about how the environment, biodiversity, and functioning were linked. We used structural equation modelling to connect fire frequency, soil fertility, exchangeable Al, water availability, functional diversity of woody plants, tree density, tree height, and litter decomposition rates in a causal chain. We found significant effects of soil nutrients, water availability, and Al on functional diversity and litter decomposition. Fire did not have a significant direct effect on functional diversity or litter decomposition. However, fire was connected to both variables through soil fertility. Functional diversity did not influence rates of litter decomposition. The mediated effects that emerged from pairwise interactions are encouraging not only for predicting the functional consequences of changes in environmental variables and biodiversity, but also to caution against predictions based on only environmental or only biodiversity change.     

A 3D doubly sensitivity enhanced X-filtered TOCSY-TOCSY experiment

We present a 3D double sensitivity enhanced X-filtered homonuclear TOCSY-TOCSY experiment for the assignment of unlabeled molecules complexed to labeled protein- or nucleic acid-domains. The resulting spectrum is clean, can be measured in a reasonable amount of time and allows for increased resolution of overlapping resonances when compared to 2D methods. The 3D X-filtered TOCSY-TOCSY allows for assignment in cases where the size or the composition of the unlabeled molecule results in a high degree of overlap.