Secreted glycoprotein from Live Zaire ebolavirus-infected cultures: preparation, structural and biophysical characterization, and thermodynamic stability.

Milligram quantities of Zaire ebolavirus nonstructural, secreted glycoprotein (sGP) were purified to homogeneity, and this preparation was characterized by an array of biophysical and biochemical experiments. Mass-spectrometry analysis revealed sGP posttranslational modifications and regions susceptible to limited proteolysis. In solution, sGP has an absolute molar mass of 103 kDa, is monodisperse, and folds into a predominantly beta -sheet conformation with a distinct tertiary structure. sGP appears to have a unique free-energy landscape that facilitates reversible folding and a strong propensity for disulfide-linked dimeric quaternary structure under a wide range of conditions; the low apparent free energy of conformation transition of sGP ( Delta G=1.7+/-0.1 kcal/mol) suggests that the molecule is well suited as a thermodynamically facile switch, which would allow it to report on relatively subtle changes in milieu. In addition, a conformational transition at 37 degrees C was detected in thermal denaturing experiments. On the basis of biophysical and biochemical considerations alone, we propose that the property of being a thermodynamically facile switch is an important clue to reveal sGP functionality.

Development, characterization and immunogenicity of a multi-stage, multi-valent Plasmodium falciparum vaccine antigen (FALVAC-1A) expressed in Escherichia coli.

A synthetic multistage, multi-epitope Plasmodium falciparum malaria antigen (FALVAC-1A) was designed and evaluated in silico, and then the gene was constructed and expressed in Escherichia coli. The FALVAC-1A protein was purified by inclusion body isolation, followed by affinity and ion exchange chromatography. Although FALVAC-1A was a synthetic antigen, it folded to a specific, but as yet incompletely defined, molecular conformation that was stable and comparable from lot to lot. When formulated with four different adjuvants, FALVAC-1A was highly immunogenic in rabbits, inducing not only ELISA reactivity to the cognate antigen and most of its component epitopes, but also in vitro activity against P. falciparum parasites as demonstrated by inhibition of sporozoite invasion, antibody dependent cellular inhibition and the immunofluorescence assay.

Acute morphine exposure potentiates the development of HSV-1-induced encephalitis.

A devastating consequence of HSV-1 infection is development of HSV-1-induced encephalitis (HSVE). While only a minority of individuals infected with HSV-1 experiences HSVE, clearly defined variables that consistently predict development of the disease remain to be elucidated. The current study examined the effects of a single dose of morphine prior to infection with HSV-1 on the development of HSVE in BALB/cByJ mice. Acute morphine exposure was observed to potentiate the development of HSVE in HSV-1 infected mice. The present data implicate a potential role for the blood-brain barrier in the development of HSVE in morphine-treated mice.

Disulfide bond assignment of the Ebola virus secreted glycoprotein SGP.

The non-structural glycoprotein (SGP) of Ebola virus (EboV) is secreted in large amounts from infected cells as a disulfide-linked homodimer. In this communication, highly purified SGP, derived from Vero E6 cultures infected with the Zaire species of EboV, was used to determine the correct localization of inter- and intrachain disulfide bonds. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of proteolytic cleavage fragments indicates that all cysteines (six per monomeric unit) form unique disulfide bonds. Monomers of the SGP homodimer are joined in a parallel manner by two intersubunit disulfide bonds formed between paired N-terminal and C-terminal cysteines (C53-C53' and C306-C306'). The remaining cysteines are involved in intrachain disulfide bonding (paired as C108-C135 and C121-C147), which resembles the disulfide bond topology of fibronectin type II domains. The findings presented here provide the foundation for future studies aimed at defining the structural and functional properties of SGP.

Redundant roles for histone H3 N-terminal lysine residues in subtelomeric gene repression in Saccharomyces cerevisiae.

The transcription of genes located in subtelomeric regions of yeast chromosomes is repressed relative to the rest of the genome. This repression requires wild-type nucleosome levels but not the telomere silencing factors Sir2, Sir3, Sir4, and Rap1. Subtelomeric heterochromatin is characterized by the absence of acetylation or methylation of histone H3 lysine residues, but it is not known whether histone H3 hypoacetylation or hypomethylation is a prerequisite for the establishment of subtelomeric heterochromatin. We have systematically mutated the N-terminal tails of histone H3 and H4 in Saccharomyces cerevisiae and characterized the effects each mutant has on genome-wide expression. Our results show that subtelomeric transcriptional repression is dependent on the histone H3 N-terminal domain, but not the histone H4 N-terminal domain. Mutating lysine-4, lysine-9, lysine-14, lysine-18, lysine-23, and lysine-27 to glycine in histone H3 is also sufficient to significantly reduce subtelomeric gene repression. Individual histone H3 lysine mutations, however, have little effect on subtelomeric gene repression or genome-wide expression, indicating that these six lysine residues have redundant functions. We propose that acetylation and methylation of histone H3 N-terminal lysine residues act as redundant mechanisms to demarcate regions of euchromatin from heterochromatin.

Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence.

The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M(-1) s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg(2+) ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg(2+) or Ca(2+), while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.

Citrus pulp for cattle.

Citrus pulp is classified as an energy concentrate by-product feed. Citrus by-products fed to beef cattle include citrus molasses, citrus meal, wet citrus pulp, dried citrus pulp, and pelleted citrus pulp; however, in current production systems, pulp (wet, dry, and pelleted) is the only by-product commonly used. Citrus pulp production in the United States is limited to specific subtropical regions, of which south central Florida remains the largest with additional production in California and Texas.

Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease.

Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 A of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 10(3)-10(5)-fold compared to that of wild-type EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 A from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small alpha-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.