Neutron Reflectometry Study of the Conformation of HIV Nef Bound to Lipid Membranes

Nef is an HIV-1 accessory protein that directly contributes to AIDS progression. Nef is myristoylated on the N-terminus, associates with membranes, and may undergo a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangement enables membrane-associated Nef to interact with cellular proteins. Despite its medical relevance, to our knowledge there is no direct information about the conformation of membrane-bound Nef. In this work, we used neutron reflection to reveal what we believe are the first details of the conformation of membrane-bound Nef. The conformation of Nef was probed upon binding to Langmuir monolayers through the interaction of an N-terminal His tag with a synthetic metal-chelating lipid, which models one of the possible limiting cases for myr-Nef. The data indicate that residues are inserted into the lipid headgroups during interaction, and that the core domain lies directly against the lipid headgroups, with a thickness of ∼40 Å. Binding of Nef through the N-terminal His tag apparently facilitates insertion of residues, as no insertion occurred upon binding of Nef through weak electrostatic interactions in the absence of the specific interaction through the His tag.     

Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses,Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.     

Engineering of a high‐throughput screening system to identify cellulosic biomass,pretreatments, and enzyme formulations that enhance sugar release

The recalcitrance of cellulosic biomass, the only abundant, sustainable feedstock for making liquid fuels, is a primary obstacle to low cost biological processing, and development of more easily converted plants and more effective enzymes would be of great benefit. Because no single parameter describes recalcitrance, superior variants can only be identified by measuring sugar release from plants subjected to pretreatment and enzymatic hydrolysis. However, genetic modifications of plants coupled with molecular engineering of deconstruction proteins and definition of pretreatment conditions create a very large sample set, and previous methods for biomass pretreatment at elevated temperatures and pressures prevented use of a fully integrated high‐throughput (HTP) screening pipeline. Herein, we report on the engineering of a novel HTP pretreatment system employing a 96 well‐plate format that withstands extreme pretreatment conditions for rapid screening of biomass–enzyme‐pretreatment combinations. This includes the development of new approaches to steam heating and water quenching the system that result in much faster heat up and cool down than previously possible and show consistent temperature histories across the multiwell plate. Coupled pretreatment and enzymatic hydrolysis performance of the well plate pretreatment system is shown to be consistent among the many wells in the device and also with performance of conventional tubular reactors. Biotechnol. Bioeng. 2010; 105: 231–238. © 2009 Wiley Periodicals, Inc.     

The Drosophila bag of marbles Gene Interacts Genetically with Wolbachia and Shows Female-Specific Effects of Divergence

Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. Here we show that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. We further find that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. We propose that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, we suggest that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes.     

The essential oils of chamaebatiaria millefolium, chamabatia australis, and chamaebatia foliolosa (Rosaceae) and comments on “Chamaebatiaria Multiflorium” and “Chamaebatiaria nelleae” as medicinal plants

The essential oil ofChamaebatiaria millefolium from California is dominated by 24.90 ± 4.46% camphor, 17.36 ± 4.23% borneol, 11.17 ± 4.26% camphene, and 10.95 ± 4.59% α-pinene. The essential oil ofChamaebatia australis from California is dominated by no constituent greater than 10% but contains 7.07 ± 0.97% δ-cadinene, 6.84 ± 1.47% terpinen-4-ol, and 5.46 ± 1.50% linalool. The essential oil ofChamaebatia foliolosa from California is dominated by 12.90 ± 3.67% unidentified sesquiterpene alcohol and 15.96 ± 6.61% viridiflorol. No scientific literature exists to substantiate the medicinal claims for “Chamae Rose.” Neither Chamaebatiaria multiflorium norChamabatiaria nelleae exist in the scientific literature.     

Receptor tyrosine kinase-G-protein coupled receptor complex signaling in mammalian cells

We describe here formation of a novel functional signaling complex between RTK and GPCRπ. This permits the use of activated G-protein subunits by the RTK in response to growth factor and that are made available by the constitutive activity of the GPCR or by binding of ligand to the latter. Moreover, β-arrestin associates with the receptor complex and participates in growth factor-dependent recruitment of c-Src, whereupon the kinase is activated by Gβγ subunits. This enables signal relay to down-stream effectors such as p42/p44 mitogen-activated protein kinases. The novel RTK–GPCR complex is involved in regulating important cellular responses, such as growth and cell migration, and dysfunction of this complex might play a significant role in hyperplasic disease states.