Silicateins, the major biosilica forming enzymes present in demosponges: protein analysis and phylogenetic relationship

Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with pI's of 5.4, of 5.2, of 4.9 and of 4.7. Surprisingly silicatein not only mediates polymerization of silicate, but also displays proteolytic activity which is specific for cathepsin L enzymes, thus underscoring the high relationship of the silicateins to cathepsin L. The cDNAs from L. baicalensis for silicatein and cathepsin L, as well as the respective genes, were cloned. It was found that the five introns present in the sponge genes are highly conserved up to human cathepsin L. This analysis has been completed by sequencing of two silicatein genes (both for silicatein-alpha and -beta) and of cathepsin L from another demosponge, Suberites domuncula. A comprehensive phylogenetic analysis with these new sequences shed new light upon the evolution of cathepsin L and silicatein families which occurred at the base of the metazoan phyla. It is concluded, that in parallel with the emergence of these enzymes at first the number of introns increased, especially in the coding region of the mature enzyme. Later in evolution the number of introns decreased again. We postulate that modification of the catalytic triad, especially of its first amino acid, is a suitable target for a chemical modulation of enzyme function of the silicateins/cathepsin L.     

Seasonal habitat use of three predatory fishes in a freshwater ecosystem

Hydrobiologia - To understand the spatiotemporal overlap in the habitat use of sympatric predators, we studied longitudinal activity and reservoir section and depth use of pike (Esox lucius),...     

Voltammetric studies of glutathione transfer across arrays of liquid–liquid microinterfaces for sensing applications

Amino Acids - The simple and facilitated transfer of tripeptide glutathione across the water/2-nitrophenyl octhyl ether interface was studied via cyclic voltammetry at interface between two...     

Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects

Nonenzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.     

Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging

Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via 'over-clocking' of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in ～24 h.     

An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore

Commonly used monomeric blue fluorescent proteins suffer from moderate brightness. The brightest of them, mTagBFP, has a notably low chemical stability over time. Prolonged incubation of mTagBFP leads to its transition from a blue fluorescent state with absorbance at 401 nm to a non-fluorescent state with absorbance at 330 nm. Here, we have determined the chemical structure of the degraded product of the blue mTagBFP-like chromophore. On the basis of mTagBFP we have developed an improved variant, named mTagBFP2. mTagBFP2 exhibits 2-fold greater chemical stability and substantially higher brightness in live cells than mTagBFP. mTagBFP2 is also 1.2-fold and 1.7-fold more photostable than mTagBFP in widefield and confocal microscopy setups, respectively. mTagBFP2 maintains all other beneficial properties of the parental mTagBFP including the high pH stability and fast chromophore formation. The enhanced photostability and chromophore chemical stability of mTagBFP2 make it a superior protein tag. mTagBFP2 performs well in the numerous protein fusions and surpasses mTagBFP as a donor in Förster resonance energy transfer with several green fluorescent protein acceptors.     

Gap Junctions and Epileptic Seizures – Two Sides of the Same Coin?

Electrical synapses (gap junctions) play a pivotal role in the synchronization of neuronal ensembles which also makes them likely agonists of pathological brain activity. Although large body of experimental data and theoretical considerations indicate that coupling neurons by electrical synapses promotes synchronous activity (and thus is potentially epileptogenic), some recent evidence questions the hypothesis of gap junctions being among purely epileptogenic factors. In particular, an expression of inter-neuronal gap junctions is often found to be higher after the experimentally induced seizures than before. Here we used a computational modeling approach to address the role of neuronal gap junctions in shaping the stability of a network to perturbations that are often associated with the onset of epileptic seizures. We show that under some circumstances, the addition of gap junctions can increase the dynamical stability of a network and thus suppress the collective electrical activity associated with seizures. This implies that the experimentally observed post-seizure additions of gap junctions could serve to prevent further escalations, suggesting furthermore that they are a consequence of an adaptive response of the neuronal network to the pathological activity. However, if the seizures are strong and persistent, our model predicts the existence of a critical tipping point after which additional gap junctions no longer suppress but strongly facilitate the escalation of epileptic seizures. Our results thus reveal a complex role of electrical coupling in relation to epileptiform events. Which dynamic scenario (seizure suppression or seizure escalation) is ultimately adopted by the network depends critically on the strength and duration of seizures, in turn emphasizing the importance of temporal and causal aspects when linking gap junctions with epilepsy.     

Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) as a potential mycoparasite on Sphaerotheca fuliginea (Ascomycotina: Erysiphales)

Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed.P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M.Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.     

McRAPD as a new approach to rapid and accurate identification of pathogenic yeasts

Despite advances in antifungal prophylaxis and therapy, morbidity and mortality incurred by yeasts remain a significant burden. As pathogenic yeast species vary in their susceptibilities to antifungal agents, clinical microbiology laboratories face an important challenge to identify them rapidly and accurately. Although a vast array of phenotyping and genotyping methods has been developed, these are either unable to cover the whole spectrum of potential yeast pathogens or can do this only in a rather costly or laborious way. Random amplified polymorphic DNA (RAPD) fingerprinting was repeatedly demonstrated to be a convenient tool for species identification in pathogenic yeasts. However, its wider acceptance has been limited mainly due to special expertise and software needed for analysis and comparison of the resulting banding patterns. Based on a pilot study, we demonstrate here that a simple and rapid melting curve analysis of RAPD products can provide data for identification of five of the most medically important Candida species. We have termed this new approach melting curve of random amplified polymorphic DNA (McRAPD) to emphasize its rapidity and potential for automation, highly desirable features for a routine laboratory test.