Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA‐cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS‐PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg²⁺, Mn²⁺, Ni²⁺, Ca²⁺, Zn²⁺, and Co²⁺ was individual for each MLCh preparation. K⁺ and Na⁺ inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis–Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (K_m = 3–9 nM) and demonstrating high k_cat values (3.4–6.9 min^?1). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular cloning and characterization of a novel anti-TLR9 intrabody

Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.     

The genus Aleurodiscus s.l. (Stereaceae, Russulales) in the Patagonian Andes

The genus Aleurodiscus s.l. in Patagonia is revised and ten species are reported, commented on, and illustrated. Aleurodiscus bernicchiae, A. corticola, A. hallenbergii, A. quilae, and A. stratosus, are proposed as new. Aleurodiscus parmuliformis is recorded for the first time out of New Zealand. A key to the species of Aleurodiscus from Patagonia is included.     

Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation of D-homo lactones

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer-Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6β,11α-dihydroxyprogesterone or 17β-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5β-dihydrotestosterone. The experiments have demonstrated that the Baeyer-Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer-Villiger oxidation, also underwent reduction to 17β-alcohols; activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) has significant impact on the amount of the formed ring-D δ-lactone.     

Loss of Rpt5 protein interactions with the core particle and Nas2 protein causes the formation of faulty proteasomes that are inhibited by Ecm29 protein

The proteasome is a large and complex protease formed by 66 polypeptides. The assembly of the proteasome is assisted by at least nine chaperones. One of these chaperones, Nas2/p27, binds to the C-terminal region of the AAA-ATPase Rpt5. We report here that the tail of Rpt5 provides two functions. First, it facilitates the previously reported interaction with the proteasome core particle (CP). Second, it is essential for the interaction with Nas2. Deletion of the C-terminal amino acid of Rpt5 disrupts the CP interaction, but not the binding to Nas2. The latter is surprising considering Nas2 contains a PDZ domain, which is often involved in binding to C termini. Interestingly, deletion of the last three amino acids interferes with both functions. The disruption of the Rpt5-CP interactions gave distinct phenotypes different from disruption of the Nas2-Rpt5 interaction. Additionally, proteasomes purified from a Saccharomyces cerevisiae rpt5-Δ3 strain show a strong enrichment of Ecm29. The function of Ecm29, a proteasome-associated protein, is not well understood. Our data show that Ecm29 can inhibit proteasomes, because our Ecm29-containing proteasomes have reduced suc-LLVY-AMC hydrolytic activity. Consistent with this apparent role as negative regulator, the deletion of ECM29 rescues the phenotypes of rpt5-Δ3 and nas2Δ in an hsm3Δ background. In sum, the interactions facilitated by the tail of Rpt5 act synergistically to minimize the formation of faulty proteasomes, thereby preventing recognition and inhibition by Ecm29.     

Prey sex pheromone as kairomone for a new group of predators (Coleoptera: Dasytidae, Aplocnemus spp.) of pine bast scales

During the last decades, an increasing number of predators were found to use specific prey pheromones as chemical cues. Beyond its ecological relevance, this knowledge has practical applications on insect conservation and pest control. In this study, we present first evidence that two species of the family Dasytidae (Coleoptera) Aplocnemus brevis Rosenhauer and A. raymondi Sainte-Claire Deville use the sex pheromone of the pine bast scale Matsucoccus feytaudi Ducasse (Hemiptera: Matsucoccidae) as kairomone to locate this prey. The feeding habits and biology of Aplocnemus species are practically unknown. In the laboratory, the adults of Aplocnemus sp. accepted M. feytaudi egg masses as food source as well as other diets. Females represented more than 90% of Aplocnemus sp. attracted to the pheromone lures. We believe that females use this olfactory cue to locate suitable places for oviposition and that larvae are the predators of Matsucoccus. This study further demonstrates that the response to the kairomone elicited short prey searching times, about 23% of the individuals appeared less than 12 min after lure exposure, being consistent with the hypothesis of prey specialization. Habitat and geographical distribution predict an ancestral association of A. brevis with M. feytaudi and of A. raymondi with M. pini. Nevertheless, a recent prey shift of A. raymondi to the invasive M. feytaudi in Corsica is in progress.     

Molecular rearrangements involved in the capsid shell maturation of bacteriophage T7

Maturation of dsDNA bacteriophages involves assembling the virus prohead from a limited set of structural components followed by rearrangements required for the stability that is necessary for infecting a host under challenging environmental conditions. Here, we determine the mature capsid structure of T7 at 1 nm resolution by cryo-electron microscopy and compare it with the prohead to reveal the molecular basis of T7 shell maturation. The mature capsid presents an expanded and thinner shell, with a drastic rearrangement of the major protein monomers that increases in their interacting surfaces, in turn resulting in a new bonding lattice. The rearrangements include tilting, in-plane rotation, and radial expansion of the subunits, as well as a relative bending of the A- and P-domains of each subunit. The unique features of this shell transformation, which does not employ the accessory proteins, inserted domains, or molecular interactions observed in other phages, suggest a simple capsid assembling strategy that may have appeared early in the evolution of these viruses.     

Cumulative effects of catch crops on nitrogen uptake, leaching and net mineralization

Catch crops can effectively decrease nitrate leaching in arable cropping systems but their long-term impacts on nitrogen mineralization are not well known. This study quantified the effects of continuous catch crops on net N mineralization, crop N uptake, crop N use efficiency and N leaching in three long-term (13?C17?years) field experiments in northern France. Mustard was grown every year at Boigneville, radish every year at Thibie and ryegrass every 2?years at Kerlavic. The mean N content of catch crop residues at these sites was 33, 36 and 35?kg ha^?1 yr^?1 and their mean C:N ratio was 13, 17 and 28, respectively. Net mineralization was calculated with a mass balance of soil mineral N using measured inputs and outputs. Catch crop establishment enhanced annual mineralization by on average 26, 18 and 9?kg N ha^?1 yr^?1 respectively during the 13?C17?year period. The difference in mineralization rate between catch crop and control treatments (extra mineralization) was positive from the first year at Boigneville, whereas it was negative or nil during the first 3?C5?years at Thibie and Kerlavic. At the latter sites, the extra mineralization rate increased significantly over time at a rate of 2.0 and 2.6?kg N ha^?1 yr^?2. At the end of the experiment, cumulative extra mineralization represented 72%, 60% and 23% of the total N added by catch crop residues at Boigneville, Thibie and Kerlavic, respectively. Repeated catch crops significantly increased N uptake and N use efficiency by main crops at Kerlavic and Thibie, but not at Boigneville. The efficiency of catch crops in reducing N leaching persisted over the years at all sites. A model simulating C and N dynamics during catch crop decomposition was able to reproduce the pattern of extra N mineralization kinetics with the various catch crop species, but underestimated the range of variation between sites. Better predictions were obtained when C or N inputs due to catch crops were increased by 10?C57%, suggesting that the actual inputs could be markedly greater than those measured in catch crop residues. According to the model, the C:N ratio of catch crop residues largely explained differences in mineralization due to different catch crop species.     

Effective immobilizing doses of medetomidine-ketamine in free-ranging, wild Norwegian reindeer (Rangifer tarandus tarandus)

Combinations of medetomidine and ketamine were evaluated in free-ranging, wild Norwegian reindeer (Rangifer tarandus tarandus) as part of a reintroduction program in southwestern Norway in November 1995 and November 1996. The drugs were administered by dart from a helicopter. The mean (SD) effective immobilizing doses for 29 adults (8 males, 21 females) were 0.21 (0.04) mg medetomidine/kg and 1.0 (0.2) mg ketamine/ kg based on estimated body mass. There was no significant difference in mean induction times between males and females. However, animals with optimal hits (shoulder or thigh muscles; n=16) had a significantly shorter (P<0.05) mean induction time than did animals with suboptimal hits (abdomen or flank; n=13), 5.6 (2.2) min and 11.1 (4.7) min, respectively. Inductions were calm, and immobilized animals were maintained in sternal recumbency. Clinical side effects included hypoxemia and hyperthermia in most animals. For reversal, all animals received 5 mg atipamezole per mg medetomidine, half intravenously and half intramuscularly, and the mean (SD) time to standing was 3.7 (3.6) min.