Modeling the in vitro 20S proteasome activity: the effect of PA28-alphabeta and of the sequence and length of polypeptides on the degradation kinetics

Proteasomes are fundamental for the degradation of intracellular proteins, having a key role in several important metabolic and signaling pathways, in the cell cycle and in antigen presentation. In vitro proteasomal digestion assays are widely used in molecular biology and immunology. We developed a model, ProteaMAlg (proteasome modeling algorithm) that describes the kinetics of specific protein fragments generated by 20S proteasomes in different conditions, once the substrate cleavage strengths are provided. ProteaMAlg was tested on a variety of data available in the literature as well as on new degradation experiments performed with polypeptides of different sequences and lengths. The comparison between in vitro and in silico experiments was used to quantify the effect on degradation of the sequence and the length of target polypeptides, of the presence of regulatory molecules such as PA28-αβ, and of the type of 20S proteasome (constitutive- or immunoproteasome). The model showed that the effect of the PA28 regulatory subunit results in a modification of the gating functions of the proteasome core particle. Immunoproteasome digestion experiments suggested that this form of proteasome, which is involved in generating MHC-class I epitopes, presents modified cleavage and gating activities. Our analysis improves the current understanding of the kinetics of proteasome functioning, and provides a tool to quantify and predict the effect of key parameters during in vitro digestion. ProteaMAlg is publicly available on the web (http://www.proteamalg.com).     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.     

Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 μM picloram or with 0.045, 0.45 and 4.5 μM 2,4-D, in both cases with and without 4.43 μM N⁶-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 μM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 μM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.     

Growth of Pleurotus ostreatus on wheat straw and wheat-grain-based media: biochemical aspects and preparation of mushroom inoculum

Mycelial growth, intracellular activity of proteases, laccases and β-1,3-glucanases, and cytoplasmic protein were evaluated in the vegetative phase of Pleurotus ostreatus grown on wheat straw and in wheat-grain-based media in Petri dishes and in bottles. The productivity of the wheat straw and wheat-grain-based spawn in cylindrical polyethylene bags containing 5 kg of chopped straw was also determined. We observed high activity of proteases and high content of intracellular protein in cultures grown on wheat straw. This suggests that the proteases are not secreted into the medium and that the protein is an important cellular reserve. On the contrary, cultures grown on wheat straw secreted laccases into the medium, which could be induced by this substrate. P. ostreatus grown on media prepared with a combination of wheat straw and wheat grain showed a high radial growth rate in Petri dishes and a high level of mycelial growth in bottles. The productivities of wheat straw and wheat-grain-based spawn were similar. Our results show that cheaper and more productive mushroom spawn can be prepared by developing the mycelium on wheat straw and wheat-grain-based substrates.     

Efficiency of population structures for mapping of Mendelian and imprinted quantitative trait loci in outbred pigs using variance component methods

In a simulation study different designs for a pure line pig population were compared for efficiency of mapping QTL using the variance component method. Phenotypes affected by a Mendelian QTL, a paternally expressed QTL, a maternally expressed QTL or by a QTL without an effect were simulated. In all alternative designs 960 progeny were phenotyped. Given the limited number of animals there is an optimum between the number of families and the family size. Estimation of Mendelian and parentally expressed QTL is more efficient in a design with large family sizes. Too small a number of sires should be avoided to minimize chances of sires to be non-segregating. When a large number of families is used, the number of haplotypes increases which reduces the accuracy of estimating the QTL effect and thereby reduces the power to show a significant QTL and to correctly position the QTL. Dense maps allow for smaller family size due to exploitation of LD-information. Given the different possible modes of inheritance of the QTL using 8 to16 boars, two litters per dam was optimal with respect to determining significance and correct location of the QTL for a data set consisting of 960 progeny. The variance component method combining linkage disequilibrium and linkage analysis seems to be an appropriate choice to analyze data sets which vary in marker density and which contain complex family structures.     

Biodiversity of pig breeds from China and Europe estimated from pooled DNA samples: differences in microsatellite variation between two areas of domestication

Microsatellite diversity in European and Chinese pigs was assessed using a pooled sampling method on 52 European and 46 Chinese pig populations. A Neighbor Joining analysis on genetic distances revealed that European breeds were grouped together and showed little evidence for geographic structure, although a southern European and English group could tentatively be assigned. Populations from international breeds formed breed specific clusters. The Chinese breeds formed a second major group, with the Sino-European synthetic Tia Meslan in-between the two large clusters. Within Chinese breeds, in contrast to the European pigs, a large degree of geographic structure was noted, in line with previous classification schemes for Chinese pigs that were based on morphology and geography. The Northern Chinese breeds were most similar to the European breeds. Although some overlap exists, Chinese breeds showed a higher average degree of heterozygosity and genetic distance compared to European ones. Between breed diversity was even more pronounced and was the highest in the Central Chinese pigs, reflecting the geographically central position in China. Comparing correlations between genetic distance and heterozygosity revealed that China and Europe represent different domestication or breed formation processes. A likely cause is a more diverse wild boar population in Asia, but various other possible contributing factors are discussed.     

Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC

The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.     

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

Background  

Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation.     

A Xenopus Dbf4 homolog is required for Cdc7 chromatin binding and DNA replication

Background  

Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase.