Integrating multiple data sources to assess the distribution and abundance of bottlenose dolphins Tursiops truncatus in Scottish waters

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Protease‐activated receptor‐1 modulates hippocampal memory formation and synaptic plasticity

Protease‐activated receptor‐1 (PAR1) is an unusual G‐protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1?/? mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1?/? mice have deficits in hippocampus‐dependent memory. We also show that while PAR1?/? mice have normal baseline synaptic transmission at Schaffer collateral‐CA1 synapses, they exhibit severe deficits in N‐methyl‐d ‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR‐mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR‐dependent processes subserving memory formation and synaptic plasticity.     

Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.     

Temporal Characteristics of Gustatory Responses in Rat Parabrachial Neurons Vary by Stimulus and Chemosensitive Neuron Type

It has been demonstrated that temporal features of spike trains can increase the amount of information available for gustatory processing. However, the nature of these temporal characteristics and their relationship to different taste qualities and neuron types are not well-defined. The present study analyzed the time course of taste responses from parabrachial (PBN) neurons elicited by multiple applications of “sweet” (sucrose), “salty” (NaCl), “sour” (citric acid), and “bitter” (quinine and cycloheximide) stimuli in an acute preparation. Time course varied significantly by taste stimulus and best-stimulus classification. Across neurons, the ensemble code for the three electrolytes was similar initially but quinine diverged from NaCl and acid during the second 500ms of stimulation and all four qualities became distinct just after 1s. This temporal evolution was reflected in significantly broader tuning during the initial response. Metric space analyses of quality discrimination by individual neurons showed that increases in information (H) afforded by temporal factors was usually explained by differences in rate envelope, which had a greater impact during the initial 2s (22.5% increase in H) compared to the later response (9.5%). Moreover, timing had a differential impact according to cell type, with between-quality discrimination in neurons activated maximally by NaCl or citric acid most affected. Timing was also found to dramatically improve within-quality discrimination (80% increase in H) in neurons that responded optimally to bitter stimuli (B-best). Spikes from B-best neurons were also more likely to occur in bursts. These findings suggest that among PBN taste neurons, time-dependent increases in mutual information can arise from stimulus- and neuron-specific differences in response envelope during the initial dynamic period. A stable rate code predominates in later epochs.     

Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi

Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.     

P2Y receptors activate neuroprotective mechanisms in astrocytic cells

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.     

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds.

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.