Poly(ADP-ribosyl)ation of chromatin in an in-vitro poly(ADP-ribose)-turnover system.

This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.     

Characterization of cloned murine cytolytic T cell lines

Murine cytolytic T lymphocytes can be kept in continuous culture apparently indefinitely by repeated passage in a concanavalin A-induced growth promoting medium. Some of these long-term cell lines maintain their cytolytic activity. Starting from three such populations, several cloned cytolytic T cell lines were derived and subsequently subcloned one or more times. Considerable variation in the levels of cytolytic activity was observed between different subclones; some initially active subclones lost activity with prolonged culture. In addition, one of the clones appeared to progressively lose the relative specificity demonstrated during the earlier passages of the parent cell line.     

Interspecific patterns for egg and clutch sizes of African Bufonidae (Amphibia: Anura)

Little is known about reproductive trade-offs in African amphibians, but such data, particularly in the form of quantitative measurements, are a key for investigating life history evolution. Here we compile and analyze known data on African bufonids from published material and new data from preserved museum specimens, to investigate interspecific patterns of egg and clutch sizes variation. Our data is a composite of mixed sources, including ova data from dissected females and laid clutches from observations in the field. Our study shows that, as body size increases, clutch size increases but egg size decreases, and when correcting for body size, egg size is inversely correlated with clutch size. These parameter interactions however, are different for different reproductive modes. In free-swimming larval developing species, the same trends are recovered, but for lecithotrophic viviparous species no significant correlations could be recovered for clutch size and body size nor for the trade-off between clutch size and egg size, and egg size is positively related to body size. The egg size of Nimbaphrynoides occidentalis (Angel, 1943) is a clear outlier, which may be due to its matrotrophic viviparous reproduction. In addition, we observed no statistical difference between ova data collected from dissections and laid clutch data from field observations, which suggests that such a mixed dataset has utility in comparative analyses.     

Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.     

Evolution of complex modular biological networks

Biological networks have evolved to be highly functional within uncertain environments while remaining extremely adaptable. One of the main contributors to the robustness and evolvability of biological networks is believed to be their modularity of function, with modules defined as sets of genes that are strongly interconnected but whose function is separable from those of other modules. Here, we investigate the in silico evolution of modularity and robustness in complex artificial metabolic networks that encode an increasing amount of information about their environment while acquiring ubiquitous features of biological, social, and engineering networks, such as scale-free edge distribution, small-world property, and fault-tolerance. These networks evolve in environments that differ in their predictability, and allow us to study modularity from topological, information-theoretic, and gene-epistatic points of view using new tools that do not depend on any preconceived notion of modularity. We find that for our evolved complex networks as well as for the yeast protein–protein interaction network, synthetic lethal gene pairs consist mostly of redundant genes that lie close to each other and therefore within modules, while knockdown suppressor gene pairs are farther apart and often straddle modules, suggesting that knockdown rescue is mediated by alternative pathways or modules. The combination of network modularity tools together with genetic interaction data constitutes a powerful approach to study and dissect the role of modularity in the evolution and function of biological networks.     

The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions

Misfolding and aggregation of proteins containing expanded polyglutamine repeats underlie Huntington's disease and other neurodegenerative disorders. Here, we show that the hetero-oligomeric chaperonin TRiC (also known as CCT) physically interacts with polyglutamine-expanded variants of huntingtin (Htt) and effectively inhibits their aggregation. Depletion of TRiC enhances polyglutamine aggregation in yeast and mammalian cells. Conversely, overexpression of a single TRiC subunit, CCT1, is sufficient to remodel Htt-aggregate morphology in vivo and in vitro, and reduces Htt-induced toxicity in neuronal cells. Because TRiC acts during de novo protein biogenesis, this chaperonin may have an early role preventing Htt access to pathogenic conformations. Based on the specificity of the Htt-CCT1 interaction, the CCT1 substrate-binding domain may provide a versatile scaffold for therapeutic inhibitors of neurodegenerative disease.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.