Establishing an In Vivo Assay System to Identify Components Involved in Environmental RNA Interference in the Western Corn Rootworm

The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR, Diabrotica virgifera virgifera) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step in vivo assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that laccase 2 and ebony, critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with ebony being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as Dicer-2 and Argonaute-2. We also evaluated two WCR sid1- like (sil) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR.     

Optical techniques for imaging membrane topography

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Angstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.     

The PLoS community journals

2005 will see the launch of three new open access journals from the Public Library of Science - PLoS Computational Biology, PLoS Genetics, and PLoS Pathogens.     

Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom

Hyaluronidase (Hyaluronate lyase, E.C. 3.2.1.35) has been isolated from Heterometrus fulvipes scorpion venom by a combination of gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and a molecular weight of 82,000. The final preparation was purified 27-fold. The optimum pH for enzyme activity was 4.0. No loss of activity was observed up to 30 degrees C and showed a sharp decrease in activity at 50 degrees C. Heparin inhibited the enzyme activity.     

Substructure of freeze-substituted plasmodesmata

Summary The substructure of plasmodesmata in freeze-substituted tissues of developing leaves of the tobacco plant (Nicotiana tabacum L. var. Maryland Mammoth) was studied by high resolution electron microscopy and computer image enhancement techniques. Both the desmotubule wall and the inner leaflet of the plasmodesmatal plasma membrane are composed of regularly spaced electron-dense particles approximately 3 nm in diameter, presumably proteinaceous and embedded in lipid. The central rod of the desmotubule is also particulate. In plasmodesmata with central cavities, spoke-like extensions are present between the desmotubule and the plasma membrane in the central cavity region. The space between the desmotubule and the plasma membrane appears to be the major pathway for intercellular transport through plasmodesmata. This pathway may be tortuous and its dimensions could be regulated by interactions between desmotubule and plasma membrane particles.Abbreviations ER endoplasmic reticulum - PJF propane jet freezing - HPF high pressure freezing - CRT cathode ray tube - IP₃ inositoltrisphosphate     

Proposed structure of putative glucose channel in GLUT1 facilitative glucose transporter.

A family of structurally related intrinsic membrane proteins (facilitative glucose transporters) catalyzes the movement of glucose across the plasma membrane of animal cells. Evidence indicates that these proteins show a common structural motif where approximately 50% of the mass is embedded in lipid bilayer (transmembrane domain) in 12 alpha-helices (transmembrane helices; TMHs) and accommodates a water-filled channel for substrate passage (glucose channel) whose tertiary structure is currently unknown. Using recent advances in protein structure prediction algorithms we proposed here two three-dimensional structural models for the transmembrane glucose channel of GLUT1 glucose transporter. Our models emphasize the physical dimension and water accessibility of the channel, loop lengths between TMHs, the macrodipole orientation in four-helix bundle motif, and helix packing energy. Our models predict that five TMHs, either TMHs 3, 4, 7, 8, 11 (Model 1) or TMHs 2, 5, 11, 8, 7 (Model 2), line the channel, and the remaining TMHs surround these channel-lining TMHs. We discuss how our models are compatible with the experimental data obtained with this protein, and how they can be used in designing new biochemical and molecular biological experiments in elucidation of the structural basis of this important protein function.     

Analysis of temperature factor distribution in high-resolution protein structures.

The temperature factors obtained from X-ray refinement of proteins at high resolution show large variations from one structure to another. However, the B-values expressed in units of standard deviation about their mean value (B'-factor) at the C alpha atoms show remarkably characteristic frequency distribution. In all of the 110 proteins examined in this study, the frequency distribution exhibited a bimodal distribution. The peaks in the B'-factor frequency distribution occur at -1.1 and 0.4 for a bin size of 0.5. The peak at lower temperature factor corresponds largely to buried residues, whereas the peak at larger value corresponds to exposed residues. The distribution could be accurately described as a superposition of two Gaussian functions. The parameters describing the distribution are therefore characteristic of protein structures. The frequency distribution for a given amino acid over all the proteins also shows a similar bimodal distribution, although the areas under the two Gaussians differ from one amino acid to another. The area under the frequency distribution curve for any interval in B'-factor represents the propensity of the amino acid to occur in that interval. This propensity is related both to the hydrophilicity/hydrophobicity of the residue and the tendency of the residue to impose a different degree of rigidity on the polypeptide chain. The frequency distribution of stretches of high B'-factors departs appreciably from that expected for a random distribution. The correlation in the B-values of sequentially proximal residues is probably responsible for the bimodal distribution.     

Modulation of phosphatidylcholine synthesis in vitro. Inhibition of diacylglycerol cholinephosphotransferase and lysophosphatidylcholine acyltransferase by centrophenoxine and neophenoxine

1,2-Diacyl-sn-glycerol : CDPcholine cholinephosphotransferase (EC 2.7.8.2) and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) activities of rat liver microsomes can be inhibited by centrophenoxine (N,N-dimethylaminoethyl p-chlorophenoxyacetate). This inhibition is brought about by the intact centrophenoxine molecule rather than by the products of hydrolysis. A nonhydrolyzable ether analog of centrophenoxine was synthesized (neophenoxine; N,N-dimethylaminoethyl p-chlorophenoxyethyl ether) and proved most effective in inhibiting the two routes of phosphatidylcholine biosynthesis. While 50% inhibition of the cholinephosphotransferase was attained at 5 mM neophenoxine, 50% inhibition of the acyltransferase required 0.6 mM neophenoxine levels only. Inhibition of the cholinephosphotransferase (Ki approximately 1.5 mM) and the acyltransferase (Ki approximately 1 mM) by neophenoxine was shown to be noncompetitive. Other membrane-bound enzymes, such as glucose-6-phosphatase, monoacylglycerol lipase, alkaline phosphatase or phospholipase A2 were not affected by the inhibitors. Because of this specificity, and because of the high affinity of the microsomal membrane for such agents, centrophenoxine and neophenoxine should prove useful for controlling phosphatidylcholine synthesis and for modulating the phosphatidylcholine deacylation-reacylation cycle.