Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage

The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.     

Genetic and functional analysis of R5X4 human immunodeficiency virus type 1 envelope glycoproteins derived from two individuals homozygous for the CCR5delta32 allele

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) isolated from two HIV-1-infected CCR5delta32 homozygotes. Envs from both subjects used CCR5 and CXCR4 for entry into transfected cells. Most R5X4 Envs were lymphocyte-tropic and used CXCR4 exclusively for entry into peripheral blood mononuclear cells (PBMC), but a subset was dually lymphocyte- and macrophage-tropic and used either CCR5 or CXCR4 for entry into PBMC and monocyte-derived macrophages. The persistence of CCR5-using HIV-1 in two CCR5delta32 homozygotes suggests the conserved CCR5 binding domain of Env is highly stable and provides new mechanistic insights important for HIV-1 transmission and persistence.     

Preparation and characterization of silica gel/chitosan composite for the removal of Cu(II) and Pb(II)

Silica gel/chitosan composite (SiCS) was prepared via., sol-gel method by mixing silica gel and chitosan and cross-linked with bifunctional cross-linker glutaraldhyde. The SiCS composite was characterized using FT-IR, SEM-EDAX, XRD and BET methods. The sorption of copper and lead ions onto SiCS has been investigated. The SiCS composite was found to have excellent metal sorption capacity than the silica gel (Si) and chitosan (CS). The sorption experiments were carried out in batch mode to optimize various parameters viz., contact time, pH, initial metal ion concentration, co-ions and temperature that influence the sorption. Langmuir, Freundlich and Dubinin-Radushkevich adsorption isotherm models were applied to describe isotherm constants. Equilibrium data agreed well with the Freundlich isotherm model. Thermodynamic studies revealed that the nature of sorption is spontaneous and endothermic. The SiCS removes metals by means of adsorption and complexation. Sorption capacity of SiCS is compared with other sorbents which suggest that this composite was useful for removing copper and lead from aqueous solution.     

Introduction of functional groups onto polypropylene and polyethylene surfaces for immobilization of enzymes

Polypropylene and polyethylene surfaces are activated by introducing an active functional group through 1-fluoro-2 nitro-4-azidobenzene by UV irradiation. Horseradish peroxidase and glucose oxidase are immobilized onto the activated surfaces, simply by incubating the enzymes at 37 degrees C. When untreated surfaces are used, insignificant immobilization of the enzymes is observed.     

Color signals in human motion-selective cortex

The neural basis for the effects of color and contrast on perceived speed was examined using functional magnetic resonance imaging (fMRI). Responses to S cone (blue-yellow) and L + M cone (luminance) patterns were measured in area V1 and in the motion area MT+. The MT+ responses were quantitatively similar to perceptual speed judgments of color patterns but not to color detection measures. We also measured cortical motion responses in individuals lacking L and M cone function (S cone monochromats). The S cone monochromats have clear motion-responsive regions in the conventional MT+ position, and their contrast-response functions there have twice the responsivity of S cone contrast-response functions in normal controls. But, their responsivity is far lower than the normals' responsivity to luminance contrast. Thus, the powerful magnocellular input to MT+ is either weak or silent during photopic vision in S cone monochromats.     

Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.     

A novel kappa agonist inhibits [3H]nimodipine binding to a Ca++ channel receptor protein in rat brain

The novel kappa agonist U50-488H in vitro produced a concentration-dependent decrease (0.25-25 microM) in [3H]nimodipine binding in neuronal P2 fractions [corrected] from rat brain cortex. Kinetic analysis indicates the decrease in binding results from a reduced Bmax with no change in affinity (Kd). The kappa antagonist, MR2266, blocked the decrease in [3H]nimodipine binding to membrane fractions. At equimolar concentrations (25 microM), morphine in vitro had no effect on [3H]nimodipine binding, while U50-488H demonstrated potent inhibition. Further kinetic analysis indicates that the IC50 for U50-488H is 0.5-0.7 microM with a KI by a Dixon plot of 1.5-1.7 microM [corrected]. These results suggest that kappa opiate receptors may be coupled to dihydropyridine receptors and as a result modulate Ca++ entry and neurotransmitter release in brain neurons.     

Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-β-D-glucuronidase (heparanase)

Mammalian heparanase is an endo-β-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.     

Bacteriophage Mu: a transposing replicon

Mu DNA replication has been carried out in vitro on cellophane discs in the presence of dBUTP. If the DNA is sheared to 80 kb pieces, the Mu replicas band anomalously in CsCl gradients between hybrid and light DNA density positions. The intermediate density DNA comprises semiconservatively replicated Mu sequences, flanked by unreplicated light DNA. This and previous data are consistent with replication occurring within Mu boundaries. Both the synthesis of Mu sequences and the intermediate density DNA are abolished by protein synthesis inhibition in vivo just prior to lysis on cellophane discs. These observations indicate that at least some steps in bona fide Mu transposition-replication are being observed in vitro.