S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.     

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.     

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.     

Life shortening in mice exposed to fission neutrons and gamma rays. V. Further studies with single low doses

Data are presented on the mean after survival of female B6CF1 mice exposed to single doses of neutrons (1 to 40 rad) or gamma rays (22.5, 45, and 90 rad). For gamma-ray exposures and for neutron exposures up to 10 rad, the dose-response curves are indistinguishable from linear; higher neutron doses produce significant departures and linearity. Consequently, in these data, an upper limit of the relative biological effectiveness (RBE) exists for life shortening from all causes of death after single neutron exposures; this value is 15.0 +/- 5.1. The RBE depends on the cause of death, ranging from 2 to 5 for lymphoreticular tumors to 23-24 for lung tumors.     

Ultrastructure of insect and spider cocoon silks

Despite much interest in the extraordinary mechanical properties of silks, the structure of native silk fibers is still not fully understood. In the present study, the morphology, topography, and organization of insect and spider cocoon silks were investigated using a range of imaging methods. Field emission scanning electron microscopy was used to observe transverse and longitude structures in silk fibers subjected to tensile fracturing, freeze fracturing, or polishing. In addition, ultrathin sections of silk brins embedded in resin were examined using transmission electron microscopy. Finally, dry silk brins were examined by confocal microscopy. The results confirmed the existence of well-oriented bundles of nanofibrils in all the silks examined and gave an indication of a hierarchical construction of the brin. Observed separation of the microfibrils in fractured brins suggests that the multifibrillar structure of the silk fiber contributes to toughness by allowing dissipation of energy in the controlled propagation of cracks.     

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.     

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.     

Influence of dexamethasone phosphate upon the DNA- and the NAD-metabolism of concanavaline a stimulated T-lymphocytes

1. Glucocorticoids have a decisive function in the immune system. In this paper, special attention is paid to the DNA and the NAD metabolism in T-lymphocytes of mice stimulated by Con A under the influence of dexamethasone phosphate. 2. Nicotinamide increases the incorporation of [3H]thymidine into the DNA of T-cells in dependence on the concentration. There is a similar but less pronounced effect with 1-methylnicotinamide. 3. Dexamethasone phosphate even at 10(-9) M inhibits the incorporation of [3H]thymidine into DNA. 4. The incorporation of [3H]thymidine into the DNA is reduced after preincubation of the T-cells with 6-aminonicotinamide or with 3-acetylpyridine. 5. Dexamethasone phosphate decreases the content of NAD in the T-cells. 6. The activity of the ADPR transferase increases after addition of Con A. Presence of nicotinamide stimulates the effect of Con A on this enzyme. This is not the case with 1-methylnicotinamide. The enzyme is inhibited drastically by dexamethasone phosphate. 7. It may be concluded that the NAD-adenoribosylation metabolism is markedly influenced by the mitogen Con A and by dexamethasone phosphate.     

The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway

The heat shock protein Gp96 has been shown to induce specific immune responses. On one hand, this phenomenon is based on the specific interaction with CD91 that mediates endocytosis and results in major histocompatibility complex class I-restricted representation of the Gp96-associated peptides. On the other hand, Gp96 induces activation of professional antigen-presenting cells, resulting in the production of pro-inflammatory cytokines and up-regulation of costimulatory molecules by unknown mechanisms. In this study, we have analyzed the consequences of Gp96 interaction with cells expressing different Toll-like receptors (TLRs) and with bone marrow-derived dendritic cells from mice lacking functional TLR2 and/or TLR4 molecules. We find that the Gp96-TLR2/4 interaction results in activation of nuclear factor kappaB-driven reporter genes and mitogen- and stress-activated protein kinases and induces IkappaBalpha degradation. Bone marrow-derived dendritic cells of C3H/HeJ and more pronounced C3H/HeJ/TLR2(-/-) mice fail to respond to Gp96. Interestingly, activation of bone marrow-derived dendritic cells depends on endocytosis of Gp96 molecules. Our results provide, for the first time, the molecular basis for understanding the Gp96-mediated activation of antigen-presenting cells by describing the simultaneous stimulation of the innate and adaptive immune system. This feature explains the remarkable ability of Gp96 to induce specific immune responses against tumors and pathogens.