Identifying temporal codes in spontaneously active sensory neurons

The manner in which information is encoded in neural signals is a major issue in Neuroscience. A common distinction is between rate codes, where information in neural responses is encoded as the number of spikes within a specified time frame (encoding window), and temporal codes, where the position of spikes within the encoding window carries some or all of the information about the stimulus. One test for the existence of a temporal code in neural responses is to add artificial time jitter to each spike in the response, and then assess whether or not information in the response has been degraded. If so, temporal encoding might be inferred, on the assumption that the jitter is small enough to alter the position, but not the number, of spikes within the encoding window. Here, the effects of artificial jitter on various spike train and information metrics were derived analytically, and this theory was validated using data from afferent neurons of the turtle vestibular and paddlefish electrosensory systems, and from model neurons. We demonstrate that the jitter procedure will degrade information content even when coding is known to be entirely by rate. For this and additional reasons, we conclude that the jitter procedure by itself is not sufficient to establish the presence of a temporal code.     

Mitochondrial haplotypes and the New Zealand origin of clonal European Potamopyrgus, an invasive aquatic snail

The small aquatic snail Potamopyrgus antipodarum is an important invading species in Europe, Australia and North America. European populations are generally believed to derive from accidental introductions from New Zealand, probably dating back to the mid-19th century. We have employed mitochondrial DNA sequences to test the proposed New Zealand origin of European Potamopyrgus, and to learn more about its genealogical history. Using a 481-bp region of the 16S ribosomal RNA gene, we identified 17 distinct haplotypes among 65 snails from New Zealand. In marked contrast, only two haplotypes were found across all European samples, which cover a large geographical area. Importantly, these two haplotypes are shared with snails from the North Island of New Zealand. Due to sampling limitations we cannot rule out a South Island origin for one of the haplotypes, but our results clearly demonstrate the New Zealand origin of European populations. The marked divergence among the two European haplotypes implies the successful colonization by two distinct mitochondrial lineages, which is consistent with previous data based on nuclear markers.     

Nesting fidelity and molecular evidence for natal homing in the freshwater turtle, Graptemys kohnii

Numerous studies of sea turtle nesting ecology have revealed that females exhibit natal homing, whereby they imprint on the nesting area from which they hatch and subsequently return there to nest as adults. Because freshwater turtles comprise the majority of reptiles known to display environmental sex determination (ESD), the study of natal homing in this group may shed light on recent evolutionary models of sex allocation that are predicated on natal homing in reptiles with ESD. We examined natal homing in Graptemys kohnii, a freshwater turtle with ESD, using mitochondrial sequencing, microsatellite genotyping and mark and recapture of 290 nesting females. Females showed high fidelity to nesting areas, even after being transplanted several kilometres away. A Mantel test revealed significant genetic isolation by distance with respect to nesting locations (r=0.147; p<0.05), suggesting that related females nest in close proximity to one another. The patterns of fidelity and genotype distributions are consistent with homing at a scale that may affect population sex ratios.     

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.     

Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo

BACKGROUND: Many signals are transduced from the cell surface to the nucleus through mitogen-activated protein (MAP) kinase cascades. Activation of MAP kinase requires phosphorylation by MEK, which in turn is controlled by Raf, Mos or a group of structurally related kinases termed MEKKs. It is not understood how MEKKs are regulated by extracellular signals. In yeast, the MEKK Ste11p functions in multiple MAP kinase cascades activated in response to pheromones, high osmolarity and nutrient starvation. Genetic evidence suggests that the p21-activated protein kinase (PAK) Ste20p functions upstream of Ste11p, and Ste20p has been shown to phosphorylate Ste11p in vitro. RESULTS: Ste20p phosphorylated Ste11p on Ser302 and/or Ser306 and Thr307 in yeast, residues that are conserved in MEKKs of other organisms. Mutating these sites to non-phosphorylatable residues abolished Ste11p function, whereas changing them to aspartic acid to mimic the phosphorylated form constitutively activated Ste11p in vivo in a Ste20p-independent manner. The amino-terminal regulatory domain of Ste11p interacted with its catalytic domain, and overexpression of a small amino-terminal fragment of Ste11p was able to inhibit signaling in response to pheromones. Mutational analysis suggested that this interaction was regulated by phosphorylation and dependent on Thr596, which is located in the substrate cleft of the catalytic domain. CONCLUSIONS: Our results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase. This mechanism may serve as a paradigm for the activation of mammalian MEKKs.     

Nanoparticles functionalized with ampicillin destroy multiple-antibiotic-resistant isolates of Pseudomonas aeruginosa and Enterobacter aerogenes and methicillin-resistant Staphylococcus aureus

We show here that silver nanoparticles (AgNP) were intrinsically antibacterial, whereas gold nanoparticles (AuNP) were antimicrobial only when ampicillin was bound to their surfaces. Both AuNP and AgNP functionalized with ampicillin were effective broad-spectrum bactericides against Gram-negative and Gram-positive bacteria. Most importantly, when AuNP and AgNP were functionalized with ampicillin they became potent bactericidal agents with unique properties that subverted antibiotic resistance mechanisms of multiple-drug-resistant bacteria.     

Classification of protein profiles from antibody microarrays using heat and detergent treatment

Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.     

Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.     

Binding interactions control SNARE specificity in vivo

Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.     

Exogenous nitric oxide triggers classic ischemic preconditioning by preventing intracellular Ca2+ overload in cardiomyocytes

The involvement of nitric oxide (NO) in the late phase of ischemic preconditioning is well established. However, the role of NO as a trigger or mediator of "classic preconditioning" remains to be determined. The present study was designed to investigate the effects of NO on calcium homeostasis in cultured newborn rat cardiomyocytes in normoxia and hypoxia. We found that treatment with the NO donor, sodium nitroprusside (SNP) induced a sustained elevation of intracellular calcium level ([Ca(2+)](i)) followed by a decrease to control levels. Elevation of extracellular calcium, which generally occurs during ischemia, caused an immediate increase in [Ca(2+)](i) and arrhythmia in cultures of newborn cardiomyocytes. Treatment with SNP decreased [Ca(2+)](i) to control levels and re-established synchronized beating of cardiomyocytes. A decrease in extracellular [Na(+)], which inhibits the Na(+)/Ca(2+) exchanger, did not prevent [Ca(2+)](i) reduction by SNP. In contrast, application of thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), increased [Ca(2+)](i), and in its presence, SNP did not reduce [Ca(2+)](i), indicating that Ca(2+) reduction is achieved via activation of SERCA2a. The results obtained suggest that activation of SERCA2a by SNP increases Ca(2+) uptake into the sarcoplasmic reticulum (SR) and prevents cytosolic Ca(2+) overload, which might explain the protective effect of SNP from hypoxic damage.