Modular evolution and the origins of symmetry: reconstruction of a three-fold symmetric globular protein

The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.     

Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α₂-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α₂-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.     

Stability of nuclear RNA in mammalian cells

The kinetics of entry of [³H]adenosine into ATP, cellular RNA, and nuclear RNA of mouse L cells were determined and analyzed. A molar accumulation curve for RNA was estimated from the specific radioactivities of RNA and ATP; this curve was resolved graphically into stable and unstable components. The stability of the unstable component (mostly heterogeneous nuclear RNA) was estimated by applying first-order decay analysis. Heterogeneous, nuclear RNA decays with an apparently uniform half-life of 23 minutes, considerably greater than some previous estimates. It is synthesized at an instantaneous rate of 5.4 × 10^?2 pg/cell per minute and reaches a steady-state level of 1.8 pg/cell in the nucleus, or 7% of the total cellular RNA. Only about 2% of the heterogeneous RNA synthesized in L cells enters polysomes as messenger RNA. The implications of these values are discussed with reference to similarly determined values for sea urchin embryos.     

Partially randomized, non-blinded trial of DNA and MVA therapeutic vaccines based on hepatitis B virus surface protein for chronic HBV infection

Background

Chronic HBV infects 350 million people causing cancer and liver failure. We aimed to assess the safety and efficacy of plasmid DNA (pSG2.HBs) vaccine, followed by recombinant modified vaccinia virus Ankara (MVA.HBs), encoding the surface antigen of HBV as therapy for chronic HBV. A secondary goal was to characterize the immune responses.

Methods

Firstly 32 HBV e antigen negative (eAg^–) participants were randomly assigned to one of four groups: to receive vaccines alone, lamivudine (3TC) alone, both, or neither. Later 16 eAg⁺ volunteers in two groups received either 3TC alone or both 3TC and vaccines. Finally, 12 eAg^– and 12 eAg⁺ subjects were enrolled into higher-dose treatment groups. Healthy but chronically HBV-infected males between the ages of 15 – 25 who lived in the western part of The Gambia were eligible. Participants in some groups received 1 mg or 2 mg of pSG2.HBs intramuscularly twice followed by 5×10⁷ pfu or 1.5×10⁸ pfu of MVA.HBs intradermally at 3-weekly intervals with or without concomitant 3TC for 11–14 weeks. Intradermal rabies vaccine was administered to a negative control group. Safety was assessed clinically and biochemically. The primary measure of efficacy was a quantitative PCR assay of plasma HBV. Immunity was assessed by IFN-γ ELISpot and intracellular cytokine staining.

Results

Mild local and systemic adverse events were observed following the vaccines. A small shiny scar was observed in some cases after MVA.HBs. There were no significant changes in AST or ALT. HBeAg was lost in one participant in the higher-dose group. As expected, the 3TC therapy reduced viraemia levels during therapy, but the prime-boost vaccine regimen did not reduce the viraemia. The immune responses were variable. The majority of IFN-γ was made by antigen non-specific CD16⁺ cells (both CD3⁺ and CD3^–).

Conclusions

The vaccines were well tolerated but did not control HBV infection.

Trial Registration

ISRCTN ISRCTN67270384     

Calcium-activated DNA fragmentation in rat liver nuclei

Incubation of isolated rat liver nuclei with ATP, NAD+, and submicromolar Ca2+ concentrations resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 200 nM Ca2+, and saturation of the process was observed with 1 microM Ca2+. ATP stimulated a calmodulin-dependent nuclear Ca2+ uptake system which apparently mediated endonuclease activation. Ca2+-activated DNA fragmentation was inhibited by the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide, and was associated with poly(ADP-ribosyl)ation of nuclear protein. The characteristics of this endonuclease activity indicate that it may be responsible for the Ca2+-dependent fragmentation of DNA involved in programmed cell death (apoptosis) and in certain forms of chemically induced cell killing.