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991.
Berhanu A Wilson RL Kirkwood-Watts DL King DS Warren TK Lund SA Brown LL Krupkin AK Vandermay E Weimers W Honeychurch KM Grosenbach DW Jones KF Hruby DE 《Journal of virology》2008,82(7):3517-3529
The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop an effective smallpox vaccine for immunization of the general public. Vaccination with the current smallpox vaccine, Dryvax, produces protective immunity but may result in adverse reactions for some vaccinees. A subunit vaccine composed of protective vaccinia virus proteins should avoid the complications arising from live-virus vaccination and thus provide a safer alternative smallpox vaccine. In this study, we assessed the protective efficacy and immunogenicity of a multisubunit vaccine composed of the A27L and D8L proteins from the intracellular mature virus (IMV) form and the B5R protein from the extracellular enveloped virus (EEV) form of vaccinia virus. BALB/c mice were immunized with Escherichia coli-produced A27L, D8L, and B5R proteins in an adjuvant consisting of monophosphoryl lipid A and trehalose dicorynomycolate or in TiterMax Gold adjuvant. Following immunization, mice were either sacrificed for analysis of immune responses or lethally challenged by intranasal inoculation with vaccinia virus strain Western Reserve. We observed that three immunizations either with A27L, D8L, and B5R or with the A27L and B5R proteins alone induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Several linear B-cell epitopes within the three proteins were recognized by sera from the immunized mice. In addition, protein-specific cellular responses were detected in spleens of immunized mice by a gamma interferon enzyme-linked immunospot assay using peptides derived from each protein. Our data suggest that a subunit vaccine incorporating bacterially expressed IMV- and EEV-specific proteins can be effective in stimulating anti-vaccinia virus immune responses and providing protection against lethal virus challenge. 相似文献
992.
R. Bension has proposed that single molecules of DNA could be sequenced rapidly, in long sequential reads, by reading off the force required to pull a tightly fitting molecular ring over each base in turn using an atomic force microscope (AFM). We present molecular dynamics simulations that indicate that pulling DNA very rapidly (m/s) could generate large force peaks as each base is passed (∼1 nN) with significant differences (∼0.5 nN) between purine and pyrimidine. These speeds are six orders of magnitude faster than could be read out by a conventional AFM, and extending the calculations to accessible speeds using Kramers’ theory shows that thermal fluctuations dominate the process with the result that purine and pyrimidine cannot be distinguished with the pulling speeds attained by current AFM technology. 相似文献
993.
Jones DS Reichardt P Ford ML Edwards LJ Evavold BD 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(3):1760-1766
Current models of T cell activation focus on the kinetics of TCR-ligand interactions as the central parameter governing T cell responsiveness. However, these kinetic parameters do not adequately predict all T cell behavior, particularly the response to antagonist ligands. Recent studies have demonstrated that TCR number is a critical parameter influencing the responses of CD4(+) T cells to weak agonist ligands, and receptor density represents an important means of regulating tissue responsiveness in other receptor ligand systems. To systematically address the impact of TCR expression on CD8(+) T cell responses, mAbs to the TCR alpha-chain and T cells expressing two TCR species were used as two different methods to manipulate the number of available TCRs on P14 and OT-I transgenic T cells. Both methods of TCR reduction demonstrated that the efficacy of antagonist peptides was significantly reduced on T cells bearing low numbers of available receptors. In addition, the ability of weak agonists to induce proliferation was critically dependent on the availability of high numbers of TCRs. Therefore, in this report we show that TCR density is a major determinant of CD8(+) T cell reactivity to weak agonist and antagonist ligands but not agonist ligands. 相似文献
994.
Reproductive fitness in many animals relies upon a tight balance between the number of cells that proliferate in the germ line and the number of cells that enter meiosis and differentiate as gametes. In the Caenorhabditis elegans germ line, the GLP-1/Notch signaling pathway controls this balance between proliferation and meiotic entry. Here we describe the identification of the proteasome as an additional regulator of this balance. We show that a decrease in proteasome activity, through either genetic mutation or RNAi to core components of the proteasome, shifts this balance toward excess germ-line proliferation. We further demonstrate that there are likely two or more proteasome targets that contribute to excess germ-line proliferation when proteasome activity is reduced. One of these targets is likely a component or regulator of the Notch-signaling pathway, while the other functions on one of the two major redundant genetic pathways downstream of GLP-1/Notch signaling. We propose a model in which the proteasome degrades proteins that are necessary for proliferation as cells switch from proliferation to meiotic entry. 相似文献
995.
Anna E. Anastasi John R. Lindsay Smith John E. McGrady 《Inorganica chimica acta》2008,361(4):1079-1086
Density functional theory suggests that the formal 2-electron oxidation of sulfides, RR′S, to sulfoxides, by the model MnVO catalyst, [(TACN)MnV O(OH)2]+, proceeds in two quite distinct 1-electron steps. Transfer of the first electron is barrierless and generates a sulfur radical cation, antiferromagnetically coupled to a MnIV centre via a covalent μ-oxo bridge. The second electron-transfer step is accompanied by migration of the oxygen atom to the sulfur centre, and is rate-determining. The absence of a barrier in the first step, where a sulfur radical is formed, means that the presence of electron-donating or withdrawing substituents on the sulfide has only a minor impact on the rate of reaction. 相似文献
996.
The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ± 0.01 μM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway. 相似文献
997.
998.
Grespin ME Bonamy GM Roggero VR Cameron NG Adam LE Atchison AP Fratto VM Allison LA 《The Journal of biological chemistry》2008,283(37):25576-25588
The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm. 相似文献
999.
Yin J Cherney MM Bergmann EM Zhang J Huitema C Pettersson H Eltis LD Vederas JC James MN 《Journal of molecular biology》2006,361(4):673-686
We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase. 相似文献
1000.
The prevalence of agglutinating antibodies to Toxoplasma gondii was examined in striped skunks (Mephitis mephitis), opossums (Didelphis virginiana), and raccoons (Procyon lotor) from 8 cities in Connecticut. Ten (42%) of the 24 striped skunks, 2 of 7 (29%) opossums, and 12 of 12 (100%) raccoons were positive at dilutions of 1:50 or greater. These results suggest that T. gondii is prevalent in the environment, or prey items, or both, of these omnivores in Connecticut. 相似文献