Self-proteolysis regulation in the Bothrops jararaca venom: the metallopeptidases and their intrinsic peptidic inhibitor

Snake venom proteome variation is a well-documented phenomenon, whereas peptidome variation is still relatively unknown. We used a biological approach to explore the inhibitory activities present in the whole venom of Bothrops jararaca that prevents the venom self-proteolysis and/or digestion of the glandular tissue. Although snake venom metallopeptidases have long been known from the biochemical up to the clinical point of view, the mechanisms by which these enzymes are regulated in the reptile's venom gland remain fairly unknown. We have successfully demonstrated that there are three synergistic weak inhibitory mechanisms that are present in the crude venom that are able to abolish the metallopeptidase activity in situ, namely: (i) citrate calcium chelation; (ii) acidic pH and; (iii) enzymatic competitive inhibition by the tripeptide Pyroglutamyl-lysyl-tryptophan. Taken together, these three factors become a strong set-up that inhibits the crude venom metallopeptidase activity as well as a purified metallopeptidase from this same venom. However, this inhibition can be totally reverted by dilution into an optimal pH solution, such as the blood.     

Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.     

Activation of human alveolar macrophages via P2 receptors: coupling to intracellular Ca2+ increases and cytokine secretion

Alveolar macrophages play a crucial role in the pathogenesis of inflammatory airway diseases. By the generation and release of different inflammatory mediators they contribute to both recruitment of different leukocytes into the lung and to airway remodeling. A potent stimulus for the release of inflammatory cytokines is ATP, which mediates its cellular effects through the interaction with different membrane receptors, belonging to the P2X and P2Y families. The aim of this study was to characterize the biological properties of purinoceptors in human alveolar macrophages obtained from bronchoalveolar lavages in the context of inflammatory airway diseases. The present study is the first showing that human alveolar macrophages express mRNA for different P2 subtypes, namely P2X(1), P2X(4), P2X(5), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(13), and P2Y(14). We also showed that extracellular ATP induced Ca(2+) transients and increased IL-1beta secretion via P2X receptors. Furthermore, extracellular nucleotides inhibited production of IL-12p40 and TNF-alpha, whereas IL-6 secretion was up-regulated. In summary, our data further support the hypothesis that purinoceptors are involved in the pathogenesis of inflammatory lung diseases.     

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction

A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

Evidence of Repeated and Independent Saltational Evolution in a Peculiar Genus of Sphinx Moths (Proserpinus: Sphingidae)

Background

Saltational evolution in which a particular lineage undergoes relatively rapid, significant, and unparalleled change as compared with its closest relatives is rarely invoked as an alternative model to the dominant paradigm of gradualistic evolution. Identifying saltational events is an important first-step in assessing the importance of this discontinuous model in generating evolutionary novelty. We offer evidence for three independent instances of saltational evolution in a charismatic moth genus with only eight species.

Methodology/Principal Findings

Maximum parsimony, maximum likelihood and Bayesian search criteria offered congruent, well supported phylogenies based on 1,965 base pairs of DNA sequence using the mitochondrial gene cytochrome oxidase subunit I, and the nuclear genes elongation factor-1 alpha and wingless. Using a comparative methods approach, we examined three taxa exhibiting novelty in the form of Batesian mimicry, host plant shift, and dramatic physiological differences in light of the phylogenetic data. All three traits appear to have evolved relatively rapidly and independently in three different species of Proserpinus. Each saltational species exhibits a markedly different and discrete example of discontinuous trait evolution while remaining canalized for other typical traits shared by the rest of the genus. All three saltational taxa show insignificantly different levels of overall genetic change as compared with their congeners, implying that their divergence is targeted to particular traits and not genome-wide.

Conclusions/Significance

Such rapid evolution of novel traits in individual species suggests that the pace of evolution can be quick, dramatic, and isolated—even on the species level. These results may be applicable to other groups in which specific taxa have generated pronounced evolutionary novelty. Genetic mechanisms and methods for assessing such relatively rapid changes are postulated.     

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

The effects of membrane filters used in biopharmaceutical processes on the concentration and composition of polysorbate 20

Polysorbate 20 (PS‐20) is often included in the formulation for therapeutic proteins to reduce protein aggregation and surface adsorption. During the production process of therapeutic proteins, various membrane filters are used to filter product pools containing PS‐20. The purpose of this study is to quantify the effects of these membrane filtration processes on the concentration and composition of PS‐20. A quantitative understanding of this process provides the knowledge base for better controlling the consistency of formulation excipients in drug products. PS‐20 solutions (without protein) were filtered through either 0.2 µm sterilizing filters or membrane filters with 30 kDa MWCO. The concentration of PS‐20 was measured by a mixed‐mode chromatography method and a nuclear magnetic resonance spectroscopy (NMR) assay. The composition of PS‐20 was characterized by ¹H‐NMR and a reverse‐phase chromatography method. Non‐specific adsorption of PS‐20 on both the sterilizing filter and 30 kDa MWCO membrane filter was quantified. Composition of PS‐20 was altered after 30 kDa MWCO membrane filtration, possibly because the different interactions between heterogeneous PS‐20 components and the 30 kDa MWCO membrane were not uniform. As a result, the retentate after the 30 kDa MWCO membrane filtration step contains no POE sorbitan and increased amount of POE sorbitan di‐esters and tri‐esters. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1503–1511, 2013