Expression Cloning of a Pseudomonas Gene Encoding a Hydroxydecanoyl-Acyl Carrier Protein-Dependent UDP-GlcNAc Acyltransferase

UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159–5169, 1987). We here report the isolation of the lpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed in Escherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624–5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of P. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predicted Pseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complemented E. coli RO138, a temperature-sensitive mutant harboring lpxA2. LpxA assays of extracts of this construct indicated that it is >1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]⁻ 1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3′.     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

Distributed torsion angle grid search in high dimensions: A systematic approach to NMR structure determination

Two complementary approaches for systematic search in torsion angle space are described for the generation of all conformations of polypeptides which satisfy experimental NMR restraints, hard-sphere van der Waals radii, and rigid covalent geometry. The first procedure is based on a recursive, tree search algorithm for the examination of linear chains of torsion angles, and uses a novel treatment to propagate the search results to neighboring regions so that the structural consequences of the restraints are fully realized. The second procedure is based on a binary combination of torsion vector spaces for connected submolecules, and produces intermediate results in Cartesian space for a more robust restraint analysis. Restraints for NMR applications include bounds on torsion angles and internuclear distances, including relational and degenerate restraints involving equivalent and nonstereoassigned protons. To illustrate these methods, conformation search results are given for the tetrapeptide APGA restrained to an idealized -turn conformation, an alanine octapeptide restrained to a right-handed helical conformation, and the structured region of the peptide SYPFDV.     

The essential oils of two chemotypes of Pinus sylvestris

The essential oils of Pinus sylvestris from French Massif Central are described. Forty-six compounds have been identified by spectral methods. Two chemotypes are recognized.     

A Simulation Study Comparing Epidemic Dynamics on Exponential Random Graph and Edge-Triangle Configuration Type Contact Network Models

We compare two broad types of empirically grounded random network models in terms of their abilities to capture both network features and simulated Susceptible-Infected-Recovered (SIR) epidemic dynamics. The types of network models are exponential random graph models (ERGMs) and extensions of the configuration model. We use three kinds of empirical contact networks, chosen to provide both variety and realistic patterns of human contact: a highly clustered network, a bipartite network and a snowball sampled network of a “hidden population”. In the case of the snowball sampled network we present a novel method for fitting an edge-triangle model. In our results, ERGMs consistently capture clustering as well or better than configuration-type models, but the latter models better capture the node degree distribution. Despite the additional computational requirements to fit ERGMs to empirical networks, the use of ERGMs provides only a slight improvement in the ability of the models to recreate epidemic features of the empirical network in simulated SIR epidemics. Generally, SIR epidemic results from using configuration-type models fall between those from a random network model (i.e., an Erdős-Rényi model) and an ERGM. The addition of subgraphs of size four to edge-triangle type models does improve agreement with the empirical network for smaller densities in clustered networks. Additional subgraphs do not make a noticeable difference in our example, although we would expect the ability to model cliques to be helpful for contact networks exhibiting household structure.     

Annotating DNA Variants Is the Next Major Goal for Human Genetics

Clinical genetic testing has undergone a dramatic transformation in the past two decades. Diagnostic laboratories that previously tested for well-established disease-causing DNA variants in a handful of genes have evolved into sequencing factories identifying thousands of variants of known and unknown medical consequence. Sorting out what does and does not cause disease in our genomes is the next great challenge in making genetics a central feature of healthcare. I propose that closing the gap in our ability to interpret variation responsible for Mendelian disorders provides a grand and unprecedented opportunity for geneticists. Human geneticists are well placed to coordinate a systematic evaluation of variants in collaboration with basic scientists and clinicians. Sharing of knowledge, data, methods, and tools will aid both researchers and healthcare workers in achieving their common goal of defining the pathogenic potential of variants. Generation of variant annotations will inform genetic testing and will deepen our understanding of gene and protein function, thereby aiding the search for molecular targeted therapies.     

Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity

There are many peptides known that inhibit the entry of enveloped viruses into cells, including one peptide that is successfully being used in the clinic as a drug. In this review, we discuss the discovery, antiviral activity and mechanism of action of such peptides. While peptide entry inhibitors have been discovered by a wide variety of approaches (structure-based, accidental, intentional, rational and brute force) we show here that they share a common physical chemical property: they are at least somewhat hydrophobic and/or amphipathic and have a propensity to interact with membrane interfaces. We propose that this propensity drives a shared mechanism of action for many peptide entry inhibitors, involving direct interactions with viral and cellular membranes, as well as interactions with the complex hydrophobic protein/lipid interfaces that are exposed, at least transiently, during virus–cell fusion. By interacting simultaneously with the membrane interfaces and other critical hydrophobic surfaces, we hypothesize that peptide entry inhibitors can act by changing the physical chemistry of the membranes, and the fusion protein interfaces bridging them, and by doing so interfere with the fusion of cellular and viral membranes. Based on this idea, we propose that an approach that focuses on the interfacial hydrophobicity of putative entry inhibitors could lead to the efficient discovery of novel, broad-spectrum viral entry inhibitors. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

The 'human revolution' in lowland tropical Southeast Asia: the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo)

Recent research in Europe, Africa, and Southeast Asia suggests that we can no longer assume a direct and exclusive link between anatomically modern humans and behavioral modernity (the 'human revolution'), and assume that the presence of either one implies the presence of the other: discussions of the emergence of cultural complexity have to proceed with greater scrutiny of the evidence on a site-by-site basis to establish secure associations between the archaeology present there and the hominins who created it. This paper presents one such case study: Niah Cave in Sarawak on the island of Borneo, famous for the discovery in 1958 in the West Mouth of the Great Cave of a modern human skull, the 'Deep Skull,' controversially associated with radiocarbon dates of ca. 40,000 years before the present. A new chronostratigraphy has been developed through a re-investigation of the lithostratigraphy left by the earlier excavations, AMS-dating using three different comparative pre-treatments including ABOX of charcoal, and U-series using the Diffusion-Absorption model applied to fragments of bones from the Deep Skull itself. Stratigraphic reasons for earlier uncertainties about the antiquity of the skull are examined, and it is shown not to be an 'intrusive' artifact. It was probably excavated from fluvial-pond-desiccation deposits that accumulated episodically in a shallow basin immediately behind the cave entrance lip, in a climate that ranged from times of comparative aridity with complete desiccation, to episodes of greater surface wetness, changes attributed to regional climatic fluctuations. Vegetation outside the cave varied significantly over time, including wet lowland forest, montane forest, savannah, and grassland. The new dates and the lithostratigraphy relate the Deep Skull to evidence of episodes of human activity that range in date from ca. 46,000 to ca. 34,000 years ago. Initial investigations of sediment scorching, pollen, palynomorphs, phytoliths, plant macrofossils, and starch grains recovered from existing exposures, and of vertebrates from the current and the earlier excavations, suggest that human foraging during these times was marked by habitat-tailored hunting technologies, the collection and processing of toxic plants for consumption, and, perhaps, the use of fire at some forest-edges. The Niah evidence demonstrates the sophisticated nature of the subsistence behavior developed by modern humans to exploit the tropical environments that they encountered in Southeast Asia, including rainforest.     

Prediction model for sequence variation in the glycoprotein gene of infectious hematopoietic necrosis virus in California, U.S.A

The influence of spatio-temporal factors on genetic variation of infectious hematopoietic necrosis virus (IHNV) is an active area of research. Using host-isolate pairs collected from 1966 to 2004 for 237 IHNV isolates from California and southern Oregon, we examined genetic variation of the mid-G gene of IHNV that could be quantified across times and geographic locations. Information hypothesized to influence genetic variation was environmental and/or fish host demographic factors, viz. location (inland or coastal), year of isolation, habitat (river, lake, or hatchery), the agent factors of subgroup (LI or LII) and serotype (1, 2, or 3), and the host factors of fish age (juvenile or adult), sex (male or female), and season of spawning run (spring, fall, late fall, winter). Inverse distance weighting (IDW) was performed to create isopleth maps of the genetic distances of each subgroup. IDW maps showed that more genetic divergence was predicted for isolates found inland (for both subgroups: LI and LII) than for coastal watershed isolates. A mixed-effect beta regression with a logit link function was used to seek associations between genetic distances and hypothesized explanatory factors. The model that best described genetic distance contained the factors of location, year of isolation, and the interaction between location and year. Our model suggests that genetic distance was greater for isolates collected from 1966 to 2004 at inland locations than for isolates found in coastal watersheds during the same years. The agreement between the IDW and beta regression analyses quantitatively supports our conclusion that, during this time period, more genetic variation existed within subgroup LII in inland watersheds than within coastal LI isolates.