Genetic improvement of spinosad resistance in the biocontrol agent Orius laevigatus

BioControl - Augmentative biological control has experienced a remarkable success, particularly in protected crops. Yet integrated pest management (IPM) still requires corrective treatments to...     

Purification and characterization of a novel alkaline α-<Emphasis Type="SmallCaps">L</Emphasis>-rhamnosidase produced by <Emphasis Type="Italic">Acrostalagmus luteo albus</Emphasis>

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal nonreducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area, with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali-tolerant soil fungus from Argentina. We also present an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In the presence of rhamnose as the sole carbon source, this fungus produces a rhamnosidase with a molecular weight of 109 kDa and a pI value of 4.6, as determined by SDS–PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrofenil-α-L-rhamnopiranoside as substrate, the enzyme activity showed pH and temperature optima of 8.0 and 55°C, respectively. The enzyme exhibited Michaelis–Menten kinetics, with K _M and V _max values of 3.38 mmol l⁻¹ and 68.5 mmol l⁻¹ min⁻¹, respectively. Neither divalent cations such as Ca²⁺, Mg²⁺, Mn²⁺, and Co²⁺ nor reducing agents such as β-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn²⁺ at a concentration of 0.2 mM. This enzyme showed the capacity to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. Based on these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered a potential new biocatalyst for industrial applications.     

Characterization of dequalinium as a XIAP antagonist that targets the BIR2 domain

Inhibitor of apoptosis proteins (IAPs) regulate the activity of caspases in apoptosis. The human X chromosome-encoded IAP (XIAP) is one of the more potent members of the IAP family and it has been described as a central regulator of apoptosis. Thus, molecules that inhibit XIAP could offer therapeutic opportunities to treat unwanted apoptosis inhibition. In the present study we have applied the selective optimization of side activities (SOSA) approach to the discovery of XIAP inhibitors. In this sense, we have identified dequalinium hydrochloride (Dq) as an inhibitor of the XIAP/caspase-3 interaction both in vitro and in cellular assays.     

The evolution of mitochondrial genomes in subterranean caviomorph rodents: adaptation against a background of purifying selection

South American tuco-tucos (Ctenomys) and the related coruro (Spalacopus) are two rodent lineages that have independently colonised the subterranean niche. The energetically demanding lifestyles of these species, coupled with the hypoxic atmospheres characteristic of subterranean environments, may have altered the selective regimes on genes encoding proteins related to cellular respiration. Here, we examined the molecular evolution of 13 protein-coding genes in the mitochondrial genome of seven caviomorph rodents, including these two subterranean genera and their above-ground relatives. Using maximum-likelihood and Bayesian approaches, we estimated rates of synonymous (dS) and nonsynonymous (dN) substitutions. We found a significantly higher ω ratio (dN/dS) in subterranean groups as compared to their non-subterranean counterparts in 11 of 13 genes, although no ω ratio was larger than 1. Additionally, we applied a method based on quantitative physicochemical properties to test for positive selection. Amino acid changes implicated in radical structural or functional shifts in the protein property were found to be ubiquitous across the phylogeny, but concentrated in the subterranean lineages. Convergent changes were also found between the subterranean genera used in this study and other mammals adapted to hypoxia. The results of this study suggest a link between niche shifts and weak directional (or episodic) selection at the molecular level against a background of purifying selection.     

A case study of virilizing adrenal tumor in an adolescent female elite tennis player--insight into the use of anabolic steroids in young athletes

A 14-year-old Caucasian girl was referred to the endocrine clinic for evaluation of voice deepening, facial hirsutism, and acne starting 2 years previously. She had been a competitive tennis player since age 7 years, practicing for 4-6 hours daily. On physical examination she was noticed to have a masculine appearance with mild facial acne and moderate hirsutism. Tanner stage was 1 for breast tissue and 5 for pubic hair. Her androgen levels (testosterone, androstenedione, dehydroepiandrosterone sulfate) were extremely elevated. Adrenal ultrasonography revealed a round left 4.6 × 5.3-cm adrenal mass. Laparoscopic left adrenalectomy was performed. The histologic findings were compatible with a benign adrenocortical tumor. Postoperatively, androgen levels dropped to within the normal range. Breast development proceeded normally, menarche occurred 2 months after tumor resection, and menses has been regular since then. Muscle strength of the dominant and nondominant upper and lower extremities was measured 1 month before surgery and 1 year later, using an isokinetic dynamometer (Biodex Systems II, Biodex, Shirley, NY, USA). There was no significant decrease in overall muscle strength after removal of the virilizing tumor and the marked drop in circulating androgens. In addition, the patient maintained her age category, number 1, national tennis ranking. The results suggest that even extremely high levels of tumor-related circulating androgens had no evident effect on muscle strength and competitive performance in a female adolescent tennis player. The lack of beneficial effect on performance in adolescents, combined with the potentially hazardous side effects of anabolic steroids, suggests that teenage athletes should avoid their use.     

Trivalent PEGylated platform for the conjugation of bioactive compounds

PEGylated multivalent structures are a new class of platform for biological applications due to their biocompatibility properties. Here, we present the synthesis of a trivalent structure 1 based on poly(ethylene glycol) units (PEG) as potential synthetic multifunctional carrier molecule. To evaluate whether this PEGylated platform could be useful for the conjugation of bioactive compounds, a well-known lipopolysaccharide (LPS) inhibitor 2, developed in our laboratory, was selected to be conjugated to 1. The LPS-neutralizing activity of the resulted conjugates and precursors was established using the chromogenic Limulus amebocyte lysate (LAL) assay. The trivalent structure 1 did not show LPS-binding activity, nonconjugate LPS inhibitor 2 showed high LPS-neutralizing activity, and the trivalent conjugate 4 displayed increased LPS-neutralizing activity and a reduced toxicity profile. These results prove the efficacy of this trivalent platform as a multivalent ligand scaffold for biological applications.     

Basolateral sorting signals regulating tissue-specific polarity of heteromeric monocarboxylate transporters in epithelia

Many solute transporters are heterodimers composed of non-glycosylated catalytic and glycosylated accessory subunits. These transporters are specifically polarized to the apical or basolateral membranes of epithelia, but this polarity may vary to fulfill tissue-specific functions. To date, the mechanisms regulating the tissue-specific polarity of heteromeric transporters remain largely unknown. Here, we investigated the sorting signals that determine the polarity of three members of the proton-coupled monocarboxylate transporter (MCT) family, MCT1, MCT3 and MCT4, and their accessory subunit CD147. We show that MCT3 and MCT4 harbor strong redundant basolateral sorting signals (BLSS) in their C-terminal cytoplasmic tails that can direct fusion proteins with the apical marker p75 to the basolateral membrane. In contrast, MCT1 lacks a BLSS and its polarity is dictated by CD147, which contains a weak BLSS that can direct Tac, but not p75 to the basolateral membrane. Knockdown experiments in MDCK cells indicated that basolateral sorting of MCTs was clathrin-dependent but clathrin adaptor AP1B-independent. Our results explain the consistently basolateral localization of MCT3 and MCT4 and the variable localization of MCT1 in different epithelia. They introduce a new paradigm for the sorting of heterodimeric transporters in which a hierarchy of apical and BLSS in the catalytic and/or accessory subunits regulates their tissue-specific polarity.     

Outer membrane continuity and septosome formation between vegetative cells in the filaments of Anabaena sp. PCC 7120

Anabaena sp. PCC 7120 is a prototype filamentous nitrogen-fixing cyanobacterium, in which nitrogen fixation and photosynthesis are spatially separated. Recent molecular and cellular studies have established the importance of molecular exchange between cells in the filament, but the routes involved are still under investigation. Two current models propose either a continuous periplasm or direct connections between adjacent cells whose integrity requires the protein SepJ. We used electron tomography to analyze the ultrastructure of the septum between vegetative cells in the Anabaena filament and were able to visualize intercellular connections that we term 'SEPTOSOMES'. We observed that, whereas the existence of the septosome does not depend on the presence of SepJ, the spacing between the two plasma membranes of the septum was significantly decreased in a ΔsepJ mutant. In addition, we observed that the peptidoglycan layer of each cell enters the septum but the outer membrane does not. Thus, each cell in the filament is individually surrounded by a plasma membrane and a peptidoglycan layer, and physical cell-cell contacts are mediated by the septosome.