Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.     

Improving Woody Biomass Feedstock Logistics by Reducing Ash and Moisture Content

We examined two practical methods for reducing moisture content and/or ash content in biomass from southern pine harvests. Logging residues are a potential bioenergy feedstock, but contaminants can increase ash content during collection. We found that trommel screens can reduce ash levels in grindings from southern pine roundwood and clean chipped logging residues from 4.0 to 1.4 % and 11.9 to 6 %, respectively. Green whole-tree chips are a widely used form of forest biomass, but are 50 % moisture. Felling and transpirationally drying in-field before chipping reduced moisture from 53 to 43 % and 39 % in 4 and 8 weeks, respectively, without changing ash content (<0.7 %). Finally, we used data from the screening and drying studies in a simulation study to estimate delivered costs for whole-tree chipping and screened and unscreened grinders processing logging and clean chip residue. Whole-tree chipping provided the lowest cost option at ash content levels less than 1 %, and unscreened grinding of clean chip residue produced the least expensive option at 5 % ash.     

Multinuclear nuclear magnetic resonance studies of Na cation-stabilized complex formed by d(G-G-T-T-T-T-C-G-G) in solution. Implications for G-tetrad structures.

There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment and report on multinuclear 1H, 31P and 13C nuclear magnetic resonance studies on the d(G2T4CG2) deoxynonanucleotide with Na cation as counterion in aqueous solution at low temperature. This sequence forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation (see the paper on d(G2T5G2) describing optical and calorimetric measurements by Jin, R., Breslauer, K. J., Jones, R. A. & Gaffney, B. L. (1990), Science, 250, 543-546). The four narrow exchangeable protons detected between 11.5 and 12.0 parts per million (p.p.m.), which are common to the d(G2T4CG2) deoxynonanucleotide and the d(G2TCG2) deoxyhexanucleotide sequences, are assigned to deoxyguanosine imino protons hydrogen-bonded to carbonyl acceptor groups. These narrow imino protons are not detected for d(IGN5IG) and d(I2N5G2), where two deoxyguanosine residues are replaced by two deoxyinosine residues in the deoxynonanucleotide sequences. This implies that the 2-amino protons of deoxyguanosine must also participate in hydrogen bond formation and stabilize the structured conformation of d(G2T4CG2) in Na cation-containing solution. We have completely assigned the base and sugar H1', H2',2', H3', and H4' protons of the d(G2T4CG2) oligomer following analysis of two-dimensional nuclear Overhauser enhancement spectroscopy and two-dimensional correlated spectroscopy data sets in 0.1 M-NaCl, 10 mM-sodium phosphate, 2H2O solution at 0 degree C. The relative magnitude of the nuclear Overhauser enhancements (NOEs) between the base H8 and its own sugar H1' protons of individual deoxyguanosine residues establishes that G1 and G8 adopt syn orientations while G2 and G9 adopt anti orientations about the glycosidic bond in the d(G1-G2-T3-T4-T5-T6-C7-G8-G9) sequence in both Na and K cation-containing aqueous solution. Consequently, any structure proposed for the tetramolecular complex of d(G2T4CG2) must exhibit alternating G(syn) and G(anti) glycosidic torsion angles within each strand. The directionality and magnitude of the observed NOEs are consistent with the G(syn)-G(anti) steps adopting right-handed helical conformations in solution. We also note that the H8 protons of G1 and G8 (7.35 to 7.45 p.p.m.) in a syn alignment are shifted significantly upfield from the H8 protons of G2 and G9 (8.0 to 8.3 p.p.m.) in an anti alignment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Laughing Falcon (Herpetotheres cachinnans) Predation on Coral Snakes (Micrurus nigrocinctus)1

Laughing falcon (Herpetotheres cachinnans) predation on coral snakes (Micrurus nigrocinctus) was recorded in two incidents that illustrate previously unreported variation in predatory behavior. In the first, the falcon held a live coral snake by the posterior end for an extended period of time, rather than decapitating it immediately. In the second, the falcon left a decapitated coral snake in a tree for more than 2 h before returning to recover its prey. A variety of behavioral adaptations may protect laughing falcons from coral snake venom.     

Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases

The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.     

Influence of landscape and vegetation characteristics on herpetofaunal assemblages in Gulf Coastal Plain pine forests

Longleaf pine (Pinus palustris) savanna characterized by open-canopy, diverse herbaceous vegetation, and high amounts of bare soil once covered much of the southeastern United States Coastal Plain. The unique structural and vegetative conditions of this ecosystem support endemic reptiles and amphibians that have declined as longleaf pine forests have been lost or degraded. Private working pine (Pinus spp.) forests managed for timber production now occur throughout the southeastern United States and have replaced much of the historical longleaf pine savanna. The examination of herpetofaunal (reptile, amphibian) communities in private working loblolly pine (P. taeda) landscapes, particularly in the western Gulf Coastal Plain is lacking. Using repeated field surveys and hierarchical community occupancy models, we examined occupancy and species richness of herpetofauna across 81 sites spanning gradients of management practices, vegetative conditions, and soil composition in northwestern Louisiana, USA, 2017–2019. Young pine stands (<6 yr) exhibited structural characteristics most similar to mature longleaf pine reference sites (>30 yr), while mid-aged stands (13–26 yr) often featured closed canopy and dense midstory. Vegetation conditions varied widely depending on landscape characteristics and site-specific disturbance regimes. We documented 43 species of herpetofauna, including 9 open-pine-associated species. Occupancy of open-pine-associated herpetofauna was positively associated with open-canopy and understory conditions, and sandy soil area. Sites providing open-canopy conditions were often occupied by open-pine-associated species regardless of overstory type and disturbance method. Overall richness of herpetofauna was greatest at sites with moderate canopy cover outside of sandy soil regions. Working pine landscapes in the western Gulf Coastal Plain can support diverse herpetofaunal assemblages, including open-pine-associated species, when management practices maintain open-canopy conditions on sandy, upland soils. More broadly, our results provide insight into how forest management practices affect herpetofauna and may guide practices that can contribute to conservation value of working pine forests.