Numerical and chemical classification ofStreptosporangium and some related actinomycetes

One hundred and seventeen streptosporangia from soil were compared with marker strains of the familyStreptosporangiaceae for many phenotypic properties. The data were examined using the Jaccard, pattern and simple matching coefficients with clustering achieved using average, complete and single linkage algorithms. Particular confidence was placed in the product of the pattern, average linkage analysis given the sharp definition of aggregate groups and clusters and a combination of low test error and high cophenetic correlation values. The test strains were assigned to five aggregate groups that were equated with the generaStreptosporangium (group A),Microbispora (group B),Planobispora andPlanomonospora (Group C),Kutzneria (neéStreptosporangium viridogriseum (group D), andMicrotetraspora (group E). The streptosporangia, both isolates and marker strains, were assigned to 5 major, 7 minor and 18 single membered clusters. Representative streptosporangia examined for chemical markers were characterised by the presence ofmeso-diaminopimelic acid in whole-organism hydrolysates, complex mixtures of straight- and branched chain fatty acids, di- and tetrahydrogenated menaquinones as predominant isoprenologues, and complex polar lipid patterns containing diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and uncharacterised components. The chemical and numerical data support the taxonomic integrity of the validly described species ofStreptosporangium and suggest that the genus is markedly underspeciated.     

The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly

Apolipoprotein (apo) B, the major protein component of the atherogenic low-density lipoprotein (LDL), has a pentapartite structure, NH2-betaalpha1-beta1-alpha2-beta2-alpha3-COOH, the beta domains containing multiple amphipathic beta strands and the alpha domains containing multiple amphipathic alpha helixes. We recently reported that the first 1000 residues of human apoB-100 have sequence and amphipathic motif homologies to the lipid-pocket of lamprey lipovitellin (LV) [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416]. The lipid-pocket of LV is a small triangular space lined by three antiparallel amphipathic beta sheets, betaA, betaB, and betaD. The betaA and betaB sheets are joined together by an antiparallel alpha helical bundle, alpha domain. We proposed [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416] that formation of a LV-like lipid-pocket is necessary for lipid-transfer to apoB-containing lipoprotein particles and that this pocket is formed by association of the region of the betaalpha1 domain homologous to the betaA and betaB sheets of LV with a betaD-like amphipathic beta sheet from microsomal triglyceride transfer protein (MTP). To test this hypothesis, we generated four truncated cDNA constructs terminating at or near the juncture of the betaalpha1 and beta1 domains: Residues 1-800 (apoB:800), 1-931 (apoB:931), 1-1000 (apoB:1000), and 1-1200 (apoB:1200). Characterization of particles secreted by stable transformants of the McA-RH7777 cell line demonstrated that (i) ApoB:800, missing the betaB domain, was secreted as a lipid-poor aggregate. (ii) ApoB:931, containing most, but not all, of the betaB domain, was secreted as lipid-poor particles unassociated with MTP. (iii) ApoB:1000, containing the entire betaB domain, was secreted as a relatively lipid-rich particle associated hydrophobically with MTP. (iv) ApoB:1200, containing the betaalpha1 domain plus 200 residues of the beta1 domain, was secreted predominantly as a lipid-poor particle but also as a minor relatively lipid-rich, MTP-associated particle. We thus have captured an intermediate in apoB-containing particle assembly, a lipid transfer competent pocket formed by association of the complete betaalpha1 domain of apoB with MTP.     

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.     

Methotrexate binding causes structural and functional changes in lung cystatin

Regulation of cysteine proteinases and their inhibitors is of utmost importance in diseases like lung cancer, chronic inflammatory conditions such as asthma, emphysema, and idiopathic pulmonary fibrosis. Protease-antiprotease imbalance accelerates disease progression. In the present study, the effect of antineoplastic and antirheumatic drug methotrexate (MTX) on lung cystatin (a cysteine protease inhibitor) was studied to explore drug induced changes in functional and structural integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of MTX with goat lung cystatin (GLC-I). Stern-Volmer analysis of MTX-GLC-I system at different temperatures indicates the presence of static component in the quenching mechanism. The thermodynamic parameters ΔH? and ΔS? were -3.8 kJ/mol and 94.97 J?mol?1?K?1, respectively, indicating that both hydrogen bonds and hydrophobic interactions played a major role in the binding of MTX to GLC-I. Methotrexate (7 μM) caused complete inactivation of lung cystatin after 6 hours. The results of FT-IR spectroscopy reflect perturbation of the goat lung cystatin on interaction with MTX. Methotrexate induced loss of function change in the inhibitor could provide a rationale for the off target tissue injury caused by the drug and for the design of agents against such an injury.     

Closing in on the resting state of the Shaker K(+) channel

Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.     

Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells

Microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins. Previously, we demonstrated that the N-terminal 1,000 residues of apoB (apoB:1000) are necessary for the initiation of apoB-containing lipoprotein assembly in rat hepatoma McA-RH7777 cells and that these particles are phospholipid (PL) rich. To determine if the PL transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB:1000-containing lipoproteins, we employed microRNA-based short hairpin RNAs (miR-shRNAs) to silence Mttp gene expression in parental and apoB:1000-expressing McA-RH7777 cells. This approach led to 98% reduction in MTP protein levels in both cell types. Metabolic labeling studies demonstrated a drastic 90–95% decrease in the secretion of rat endogenous apoB100-containing lipoproteins in MTP-deficient McA-RH7777 cells compared with cells transfected with negative control miR-shRNA. A similar reduction was observed in the secretion of rat endogenous apoB48 under the experimental conditions employed. In contrast, MTP absence had no significant effect on the synthesis, lipidation, and secretion of human apoB:1000-containing particles. These results provide strong evidence in support of the concept that in McA-RH7777 cells, acquisition of PL by apoB:1000 and initiation of apoB-containing lipoprotein assembly, a process distinct from the conventional first-step assembly of HDL-sized apoB-containing particles, do not require MTP. This study indicates that, in hepatocytes, a factor(s) other than MTP mediates the formation of the PL-rich primordial apoB:1000-containing initiation complex.     

Synthesis of an Fmoc-threonine bearing core-2 glycan: A building block for PSGL-1 via Fmoc-assisted solid-phase peptide synthesis

Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing sialyl Lewis^x oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This building block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a portion of the PSGL-1 glycopeptide.     

Changing Museum Visitors’ Conceptions of Evolution

We examined whether a single visit to an evolution exhibition contributed to conceptual change in adult (n?=?30), youth, and child (n?=?34) museum visitors?? reasoning about evolution. The exhibition included seven current research projects in evolutionary science, each focused on a different organism. To frame this study, we integrated a developmental model of visitors?? understanding of evolution, which incorporates visitors?? intuitive beliefs, with a model of free-choice learning that includes personal, sociocultural, and contextual variables. Using pre- and post-measures, we assessed how visitors?? causal explanations about biological change, drawn from three reasoning patterns (evolutionary, intuitive, and creationist), were modified as a result of visiting the exhibition. Whatever their age, background beliefs, or prior intuitive reasoning patterns, visitors significantly increased their use of explanations from the evolutionary reasoning pattern across all measures and extended this reasoning across diverse organisms. Visitors also increased their use of one intuitive reasoning pattern, need-based (goal-directed) explanations, which, we argue, may be a step toward evolutionary reasoning. Nonetheless, visitors continued to use mixed reasoning (endorsing all three reasoning patterns) in explaining biological change. The personal, socio-cultural, and contextual variables were found to be related to these reasoning patterns in predictable ways. These findings are used to examine the structure of visitors?? reasoning patterns and those aspects of the exhibition that may have contributed to the gains in museum visitors?? understanding of evolution.