C-terminal region of bacterial Ku controls DNA bridging,DNA threading and recruitment of DNA ligase D for double strand breaks repair

Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.     

Nile perch and the transformation of Lake Victoria

The transformation of Lake Victoria that began in 1980 followed the population explosion of Nile perch Lates niloticus, causing the apparent extirpation of 500+ endemic haplochromine species and dramatic physico-chemical changes. Officially introduced in 1962–1963, but present earlier, the reasons for the long delay before its population exploded are discussed. The hypothesis that it occurred only after the haplochromine decline is evaluated, but haplochromines declined only after the Nile perch expansion began. The sudden eutrophication of the lake was attributed to Nile perch, but evidence of eutrophication from 1950 onwards led some researchers to conclude that it was the result of climatic changes. We conclude that the haplochromine destruction disrupted the complex food webs that existed prior to the upsurge of Nile perch. The depletion of fish biomass by Nile perch may have been the source of extra phosphorus responsible for the eutrophication of the lake. After the Nile perch explosion in 1980 the fish population came to be dominated by only three species, but fisheries productivity increased at least 10-fold. Fishing has caused demographic changes in Nile perch, which may have allowed some haplochromine species to recover. The condition of the lake appears to have stabilised since 2000, partly because the fish biomass has risen to at least 2 × 10⁶ t, replacing the ‘lost’ biomass and restoring some ecosystem functioning.     

A force plate study of avian gait

OBJECTIVE: To test the force plate as a gait analysis system for broilers and to determine how the ground reaction force (GRF) patterns change in these birds with growth and administration of analgesia. MATERIALS AND METHODS: Thirty-three male Ross 308 chicks were raised on either an ad libitum or restricted-feeding regime, and subsequently treated with carprofen or a placebo. Vertical, craniocaudal and mediolateral GRFs were measured as the birds walked across a standard force plate. RESULTS: The data were easy to collect, and peak vertical forces of an equivalent percentage of bodyweight as seen in human walking were identified. Mediolateral forces were 2-3 times greater than those demonstrated in other species. GRF patterns showed significant changes during growth, but analgesia did not have a significant effect on the speed of walking, or GRF patterns. CONCLUSIONS AND CLINICAL RELEVANCE: The force plate is a suitable research tool for recording GRFs from avian bipeds. The large mediolateral forces identify a particularly inefficient aspect of avian gait; however, the role of pain remains to be determined.     

Defining Elastic Fiber Interactions by Molecular Fishing: AN AFFINITY PURIFICATION AND MASS SPECTROMETRY APPROACH*

Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix.Mass spectrometry is emerging as a powerful approach to identify protein interaction partners in molecular complexes. We have developed an affinity purification and mass spectrometry strategy that is applicable to the analysis of molecular interactions of extracellular matrix complexes. The extracellular matrix provides structural support to tissues and profoundly influences cell survival, proliferation, migration, and phenotypic state. It is a complex multimolecular and three-dimensional milieu that comprises assembled networks of tissue-specific combinations of structural and cell-adhesive glycoproteins, proteoglycans, and cross-linking enzymes. The matrix also sequesters numerous growth factors and cytokines, thereby controlling their bioavailability. Delineating the molecular nature of the fundamental interacting networks within complex extracellular matrices is a challenging task. Here, mass spectrometry has given new insights into elastic fiber interactions.Elastic fibers are essential structural elements of the extracellular matrix of dynamic connective tissues such as blood vessels, lungs, skin, and ligaments, endowing these tissues with elastic recoil (1, 2). Their importance is emphasized by elastic fiber defects that cause severe acquired diseases such as aortic aneurysms and pulmonary emphysema and life-threatening heritable disorders such as Marfan syndrome, supravalvular stenosis, and cutis laxa. These fibers are extensive multimolecular assemblies that adopt intricate tissue-specific architectural arrangements. At the morphological level, the fibers comprise a cross-linked elastin core and an outer mantle of fibrillin microfibrils. It has proved challenging to define the composition of tissue elastic fibers biochemically. Cross-linked elastin is highly insoluble and its isolation from tissues requires extreme conditions of hot alkali, which destroys other proteins (2). The efficient extraction of tissue microfibrils requires collagenase and other proteolytic activities that may destroy associated molecules (3). Despite these difficulties, a number of associated proteins, including MAGP-1,1 βigH3, fibulins, and lysyl oxidases (LOX and LOXL (also known as LOXL1)), as well as latent TGFβ-binding proteins (LTBPs), collagen VIII, and emilin-1 have been identified in biochemical and/or colocalization studies (1).Fibrillins are very large glycoproteins (350 kDa) containing 43 calcium-binding epidermal growth factor-like domains and seven TGFβ-binding protein-like (8-cysteine) domains (4). Fibrillin-1 is the more abundant isoform; fibrillin-2 is mainly expressed during development (5, 6). Tropoelastin, the secreted soluble form of elastin, comprises alternating hydrophobic and lysine-rich cross-linking domains. LOX and LOXL are copper-dependent amine oxidases that cross-link elastin through the oxidative deamination of specific lysines (7–9). Elastin is mainly expressed and deposited early in life and undergoes very little turnover in healthy tissues (2). MAGP-1 is a microfibril-associated glycoprotein that binds fibrillin-1 and elastin (10, 11) but is not essential for elastic fiber formation (12). βigH3 was originally identified as a matrix protein, MP78/70, in tissue extracts that solubilized elastin-associated microfibrils (13, 14). Fibulin-4 and -5 play essential roles in elastic fiber formation (15, 16), most likely by regulating elastin deposition onto microfibrils (17, 18). Fibulin-2 interacts with fibrillin-1 (19) but is not essential for elastic fiber formation (20). Fibulin-1-null mice, among other symptoms, display anomalies of aortic arch arteries and hemorrhagic blood vessels, suggesting some involvement in elastic fiber biology (21). Fibulin-3 (also known as Efemp1)-deficient mice exhibit early aging and herniation associated with reduced elastic fiber integrity (22). Collagen VIII and emilin-1 also colocalize to elastic fibers (23, 24).The assembly of microfibrils and elastic fibers remains incompletely understood. We and others recently showed that assembly of the microfibril component is orchestrated by the cell surface through interactions with fibronectin and integrin receptors (25, 26). Heparan sulfate, an abundant pericellular glycosaminoglycan chain attached to syndecan and glypican proteoglycan receptors, also critically influences microfibril formation (27–29). Elastin deposition and stabilization on microfibrils require fibulins and the cross-linking enzymes LOX and/or LOXL.To obtain new insights into the molecular interactions of elastic fibers and how they are integrated into their surrounding matrix, we conducted a detailed affinity capture LC-MS/MS analysis of molecules that interact in culture specifically with four His₆-tagged recombinant human elastic fiber molecules (fibrillin-1, MAGP-1, fibulin-5, and LOX). Tropoelastin was not used as bait because of its highly adhesive nature. Our protocol proved to be an effective strategy for defining specific interactions of elastic fiber molecules in the extracellular matrix. Efficacy was demonstrated through confirmation of known interactions and validation of novel extracellular matrix protein-protein interactions. This approach further allowed us to predict secondary elastic fiber interactions, giving powerful insights into the molecular networks that sustain elastic fibers within higher order extracellular matrices.     

Affects of two cotton varieties on levels of boll weevil parasitism [Col.: Curculionidae]

The cotton plant has a profound affect on the behavior and ability of parasites to successfully attack the boll weevil,Anthonomus grandis Boheman. The most important native parasite of the boll weevil,Bracon mellitor Say, prefers to oviposit on Frego bract cotton rather than normal bract cotton. An imported parasite,Heterolaccus grandis Burks prefers ovipositions on normal bract cotton rather than Frego.     

Biochemical studies on the H-2K antigens of the MHC mutantbm1

Biochemical analysis of the H-2K-gene product from the MHC mutant strainbml and from the C57BL/6 parent strain has been carried out in order to characterize the structural differences between parent and mutant K-gene products. Based on comparative tryptic peptide mapping of the cyanogen bromide fragments from these glycoproteins, two peptide differences were localized to the CN-Ia fragment. Partial amino-acid sequence analysis revealed two alterations in the primary structure of K^bml involving substitutions of tyrosine for arginine at position 155, and tyrosine for leucine at position 156. Both of these amino-acid replacements require a minimum of two nucleotide base changes at the nucleic acid level. These changes were the only alterations noted differentiating the K^bml and K^b glycoproteins. However, because our techniques allow us to analyze only 75 to 80 percent of the extra cellular portion of H-2K^b, it is possible there are other undetected changes. Nonetheless, the biochemical data are consistent with the hypothesis that the structural alterations noted in the K^bml mutant glycoprotein are directly related to the observed immunological specificity relative to the parent K^b molecule. Peptide comparisons of the K^b molecules of two C57BL/6 sublines and of the H-2^b lymphoblastoid cell line, EL-4, disclosed no difference.     

Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: application to human immunodeficiency viral protease

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNbeta-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detection conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with apparent first-order kinetics with a Km of 37.8 +/- 8.7 microM and a kcat of 0.95 +/- 0.07 s-1. Examination of the first-order rate constant versus enzyme concentration suggested a Kd of 9.4 +/- 2.7 nM for the HIV protease monomer-dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors.     

Caspase-dependent deubiquitination of monoubiquitinated nucleosomal histone H2A induced by diverse apoptogenic stimuli.

Enzymatic deubiquitination of mono-ubiquitinated nucleosomal histone H2A (uH2A) and H2B (uH2B) is closely associated with mitotic chromatin condensation, although the function of this histone modification in cell division remains ambiguous. Here we show that rapid and extensive deubiquitination of nucleosomal uH2A occurs in Jurkat cells undergoing apoptosis initiated by anti-Fas activating antibody, staurosporine, etoposide, doxorubicin and the proteasome inhibitor, N-acetyl-leucyl-leucyl-norlucinal. These diverse apoptosis inducers also promoted the accumulation of slowly migrating, high molecular weight ubiquitinated proteins and depleted the cellular pool of unconjugated ubiquitin. In apoptotic cells, ubiquitin was cleaved from uH2A subsequent to the appearance of plasma membrane blebbing, and deubiquitination of uH2A closely coincided with the onset of nuclear pyknosis and chromatin condensation. Nucleosomal uH2A deubiquitination, poly (ADP-ribose)polymerase (PARP) cleavage and chromatin condensation were prevented in cells challenged with apoptosis inducers by pretreatment with the pan-caspase inhibitor, zVAD-fmk, or by over-expressing anti-apoptotic Bcl-xL protein. These results implicate a connection between caspase cascade activation and nucleosomal uH2A deubiquitination. Transient transfection of 293 cells with the gene encoding Ubp-M, a human deubiquitinating enzyme, promoted uH2A deubiquitination, while an inactive mutated Ubp-M enzyme did not. However, Ubp-M-promoted deubiquitination of uH2A was insufficient to initiate apoptosis in these cells. We conclude that uH2A deubiquitination is a down-stream consequence of procaspase activation and that unscheduled cleavage of ubiquitin from uH2A is a consistent feature of the execution phase of apoptosis rather than a determining or initiating apoptogenic event. Nucleosomal uH2A deubiquitination may function as a cellular sensor of stress in situations like apoptosis through which cells attempt to preserve genomic integrity.