Biomarkers of toxicity in Clarias gariepinus exposed to sublethal concentrations of polycyclic aromatic hydrocarbons

Physiological, biochemical and histological indices in Clarias gariepinus broodstock, and teratogenic indices in embryos exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene were investigated in 2014 using a static-renewal bioassay protocol. Phenanthrene (1.41 mg l^?1) was the most toxic, followed by pyrene (1.53 mg l^?1) and naphthalene (7.21 mg l^?1), based on 96 h LC50 values. Hepatosomatic indices were significantly higher in naphthalene- and pyrene-treated males compared with solvent controls, whereas fecundity in females was significantly lower by factors of 2.4 (naphthalene), 2.8 (phenanthrene) and 2.4 (pyrene), compared with controls. Catalase levels were lower in female phenanthrene-treated fish compared with controls. Histological alterations observed in PAH-treated fish include oedema, inflammatory cells, epithelial lifting and hyperplasia in the gills, vacuolation, haemosiderin pigments and sinusoidal congestion in the liver, and degenerated zona radiata in the ovary. Teratogenic effects were not observed, as evidenced by the lack of histological alterations in embryos spawned from pre-exposed broodstock. Sex-specific responses and the utility of biomarkers at cellular and individual levels of organisation are therefore demonstrated for holistic evaluations of polycyclic aromatic hydrocarbons in ecotoxicological studies.     

The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.     

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca²⁺ and Mn²⁺. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.Anthrax is an infectious disease caused by the encapsulated, spore-forming bacterium Bacillus anthracis. Systemic forms of the disease, such as inhalational anthrax, are characterized by nonspecific early symptoms, rapid progression, and lethality approaching 100% (1). The lethality of inhalational anthrax is high even with antibiotic treatment and is caused by accumulation of secreted anthrax toxin (2), which consists of three proteins as follows: protective antigen (PA),² lethal factor (LF), and edema factor. PA binds to membrane receptors, forms pore complexes, and translocates LF and edema factor into the host cell (3, 4). The PA·LF complex is known as the lethal toxin, a virulence factor with pleiotropic action that facilitates establishment of the B. anthracis infection. LF is a Zn²⁺-dependent metalloprotease related to the thermolysin family that cleaves mitogen-activated protein kinase kinases (5).Although the complete mechanism by which LF causes fatal intoxication is still unclear, inhibition of LF proteolytic activity may be an efficient means of preventing anthrax lethality. A better understanding of the LF catalytic mechanism will facilitate rational design and optimization of LF inhibitors with potential clinical applicability. Recent structural (6, 7), mechanistic (8), and in vivo studies (9, 10) of LF point to a sophisticated catalytic mechanism involving accurate recognition of multiple target substrates.Here we use substrate phage display and stopped-flow fluorimetry kinetics to examine both the substrate specificity and elementary steps of substrate processing by LF. Our data allow us to construct a working model of LF-substrate binding and cleavage.     

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples

Background  

Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C₄(RP)-LC protein separation) followed by C₁₈(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.     

Ultrastructure of yeast cell <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> after amiodarone treatment

The ultrastrcutre of Saccharomyces cerevisiae cells (wild-type and ysp2 mutant cells) was studied after amiodarone treatment. Amiodarone is used as a pharmaceutical substance for treating a number of diseases; however, it is known that amiodarone causes structural and functional disturbances in patient tissues. Here, the peculiarities of the amiodarone effect are studied in Saccharomyces cerevisiae yeast, in which amiodarone has been shown to cause apoptosis. Electron-microscopic study of yeast cells after amiodarone treatment reveals a significant increase in the number of lipid particles, which can lead to the formation of a structural complex by interacting with cell membranous organelles. Amiodarone causes the appearance of small and slightly swollen mitochondria. Chromatin displacement to the periphery of the nucleus, nuclear sectioning, and nuclear envelope disturbances are observed in the cells under these conditions. The detected changes int eh ultrastructure of the cell in Saccharomyces cerevisiae are considered to be a specific response to phospholipidosis and apoptosis caused by amiodarone.     

Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.