A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Using diffusion distances for flexible molecular shape comparison

Background  

Many molecules are flexible and undergo significant shape deformation as part of their function, and yet most existing molecular shape comparison (MSC) methods treat them as rigid bodies, which may lead to incorrect shape recognition.     

The BNIP-2 and Cdc42GAP Homology (BCH) Domain of p50RhoGAP/Cdc42GAP Sequesters RhoA from Inactivation by the Adjacent GTPase-activating Protein Domain

The BNIP-2 and Cdc42GAP homology (BCH) domain is a novel regulator for Rho GTPases, but its impact on p50-Rho GTPase-activating protein (p50RhoGAP or Cdc42GAP) in cells remains elusive. Here we show that deletion of the BCH domain from p50RhoGAP enhanced its GAP activity and caused drastic cell rounding. Introducing constitutively active RhoA or inactivating GAP domain blocked such effect, whereas replacing the BCH domain with endosome-targeting SNX3 excluded requirement of endosomal localization in regulating the GAP activity. Substitution with homologous BCH domain from Schizosaccharomyces pombe, which does not bind mammalian RhoA, also led to complete loss of suppression. Interestingly, the p50RhoGAP BCH domain only targeted RhoA, but not Cdc42 or Rac1, and it was unable to distinguish between GDP and the GTP-bound form of RhoA. Further mutagenesis revealed a RhoA-binding motif (residues 85-120), which when deleted, significantly reduced BCH inhibition on GAP-mediated cell rounding, whereas its full suppression also required an intramolecular interaction motif (residues 169-197). Therefore, BCH domain serves as a local modulator in cis to sequester RhoA from inactivation by the adjacent GAP domain, adding to a new paradigm for regulating p50RhoGAP signaling.     

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.     

MALE PEACH APHID ATTRACTION IN THE FIELD BY SEX PHEROMONES1)

Abstract Field trials by sex pheromone of aphid to trap peach aphids Myzus persicae have been carried out in 1995 and in 1996. Suitable time and the effect of ratio of two components nepetalactone and nepetalactol to apply the lure have been observed.     

Size-dependent mortality in a Neotropical savanna tree: the role of height-related adjustments in hydraulic architecture and carbon allocation

Size-related changes in hydraulic architecture, carbon allocation and gas exchange of Sclerolobium paniculatum (Leguminosae), a dominant tree species in Neotropical savannas of central Brazil (Cerrado), were investigated to assess their potential role in the dieback of tall individuals. Trees greater than ∼6-m-tall exhibited more branch damage, larger numbers of dead individuals, higher wood density, greater leaf mass per area, lower leaf area to sapwood area ratio (LA/SA), lower stomatal conductance and lower net CO₂ assimilation than small trees. Stem-specific hydraulic conductivity decreased, while leaf-specific hydraulic conductivity remained nearly constant, with increasing tree size because of lower LA/SA in larger trees. Leaves were substantially more vulnerable to embolism than stems. Large trees had lower maximum leaf hydraulic conductance ( K _leaf) than small trees and all tree sizes exhibited lower K _leaf at midday than at dawn. These size-related adjustments in hydraulic architecture and carbon allocation apparently incurred a large physiological cost: large trees received a lower return in carbon gain from their investment in stem and leaf biomass compared with small trees. Additionally, large trees may experience more severe water deficits in dry years due to lower capacity for buffering the effects of hydraulic path-length and soil water deficits.     

Comparison Study of Bone Defect Healing Effect of Raw and Processed Pyritum in Rats

To evaluate and compare the effect of raw and processed pyritum on tibial defect healing, 32 male Sprague Dawley rats were randomly divided into four groups. After tibial defect, animals were produced and grouped: sham and control group were orally administrated with distilled water (1 mL/100 g), while treatment groups were given aqueous extracts of raw and processed pyritum (1.5 g/kg) for successive 42 days. Radiographic examination showed that bone defect healing effect of the treatment groups was obviously superior compared to that of the control group. Bone mineral density of whole tibia was increased significantly after treating with pyritum. Inductively coupled plasma-optical emission spectrometry showed that the contents of Ca, P, and Mg in callus significantly increased in the treatment groups comparing with the control. Moreover, serological analysis showed that the concentration of serum phosphorus of the treatment groups significantly increased compared with that of the control group. By in vitro study, we have evaluated the effects of drug-containing serum of raw and processed pyritum on osteoblasts. It was manifested that both the drug-containing sera of raw and processed pyritum significantly increased the mRNA levels of alkaline phosphatase and collagen type I. Protein levels of phosphorylated Smad2/3 also increased. The mRNA levels of osteocalcin and transforming growth factor β (TGF-β) type I and II receptors, as well as the protein levels of TGF-β1 in the processed groups, were higher than those in the control. In summary, both raw and processed pyritum-containing sera exhibited positive effects on osteoblasts, which maybe via the TGF-β1/Smad signaling pathway. Notably, the tibia defect healing effect of pyritum was significantly enhanced after processing.     

Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.