Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

The influence of temperature on the dynamics of protein precrystallization clusters,studied by photon correlation spectroscopy

Hen-egg white lysozyme was used for studying the influence of temperature on crystallization. The reaction was initiated at variable temperatures, covering the range between 5–50 °C, and was monitored with photon correlation spectroscopy. When aggregation was induced by addition of NaCl, the clusters formed exhibited diffusion limited aggregation behavior and crystals appeared in less than two days. In contrast, (NH₄)₂SO₄ induced aggregation took place mostly in the cross-over regime. In this case, solutions either remained transparent and void of crystals or formed gels within a few weeks. In both cases the kinetics could be dynamically scaled into master curves indicating that the precrystallization formed aggregates are fractals resulting from different collision processes.     

Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.     

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) fromSilene alba cells

The peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992)Glycoconjugate J 9:191–97] was partially purified from culturedSilene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of unconjugated N-glycans in a suspension medium of culturedSilene alba cells.Abbreviations GlcNAc N-acetylglucosamine - PNGase peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase - BSA bovine serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TLC thin layer chromatography - HPAEC-PAD High pH anion exchange chromatography-pulsed amperometric detection     

Into the deep: the functionality of mesopelagic excursions by an oceanic apex predator

Comprehension of ecological processes in marine animals requires information regarding dynamic vertical habitat use. While many pelagic predators primarily associate with epipelagic waters, some species routinely dive beyond the deep scattering layer. Actuation for exploiting these aphotic habitats remains largely unknown. Recent telemetry data from oceanic whitetip sharks (Carcharhinus longimanus) in the Atlantic show a strong association with warm waters (>20°C) less than 200 m. Yet, individuals regularly exhibit excursions into the meso‐ and bathypelagic zone. In order to examine deep‐diving behavior in oceanic whitetip sharks, we physically recovered 16 pop‐up satellite archival tags and analyzed the high‐resolution depth and temperature data. Diving behavior was evaluated in the context of plausible functional behavior hypotheses including interactive behaviors, energy conservation, thermoregulation, navigation, and foraging. Mesopelagic excursions (n = 610) occurred throughout the entire migratory circuit in all individuals, with no indication of site specificity. Six depth‐versus‐time descent and ascent profiles were identified. Descent profile shapes showed little association with examined environmental variables. Contrastingly, ascent profile shapes were related to environmental factors and appear to represent unique behavioral responses to abiotic conditions present at the dive apex. However, environmental conditions may not be the sole factors influencing ascents, as ascent mode may be linked to intentional behaviors. While dive functionality remains unconfirmed, our study suggests that mesopelagic excursions relate to active foraging behavior or navigation. Dive timing, prey constituents, and dive shape support foraging as the most viable hypothesis for mesopelagic excursions, indicating that the oceanic whitetip shark may regularly survey extreme environments (deep depths, low temperatures) as a foraging strategy. At the apex of these deep‐water excursions, sharks exhibit a variable behavioral response, perhaps, indicating the presence or absence of prey.     

Lysosomal Signaling Licenses Embryonic Stem Cell Differentiation via Inactivation of Tfe3

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Observation of a porbeagle shark Lamna nasus aggregation at a North Sea oil platform

A minimum of 20 porbeagles Lamna nasus were observed circling the Alba oil platform (UK North Sea) over several days in July 2014. Although schools of fish often aggregate around oil platforms, less is known about their ability to aggregate pelagic sharks and L. nasus are rarely seen at the surface. This unusual observation provides new insights into L. nasus behaviour and habitat use and the potential role of offshore artificial structures on pelagic predators.     

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.     

Growth and Maximum Size of Tiger Sharks (Galeocerdo cuvier) in Hawaii

Tiger sharks (Galecerdo cuvier) are apex predators characterized by their broad diet, large size and rapid growth. Tiger shark maximum size is typically between 380 & 450 cm Total Length (TL), with a few individuals reaching 550 cm TL, but the maximum size of tiger sharks in Hawaii waters remains uncertain. A previous study suggested tiger sharks grow rather slowly in Hawaii compared to other regions, but this may have been an artifact of the method used to estimate growth (unvalidated vertebral ring counts) compounded by small sample size and narrow size range. Since 1993, the University of Hawaii has conducted a research program aimed at elucidating tiger shark biology, and to date 420 tiger sharks have been tagged and 50 recaptured. All recaptures were from Hawaii except a single shark recaptured off Isla Jacques Cousteau (24°13′17″N 109°52′14″W), in the southern Gulf of California (minimum distance between tag and recapture sites  =  approximately 5,000 km), after 366 days at liberty (DAL). We used these empirical mark-recapture data to estimate growth rates and maximum size for tiger sharks in Hawaii. We found that tiger sharks in Hawaii grow twice as fast as previously thought, on average reaching 340 cm TL by age 5, and attaining a maximum size of 403 cm TL. Our model indicates the fastest growing individuals attain 400 cm TL by age 5, and the largest reach a maximum size of 444 cm TL. The largest shark captured during our study was 464 cm TL but individuals >450 cm TL were extremely rare (0.005% of sharks captured). We conclude that tiger shark growth rates and maximum sizes in Hawaii are generally consistent with those in other regions, and hypothesize that a broad diet may help them to achieve this rapid growth by maximizing prey consumption rates.