Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists

The HIV-1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat-antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O-sulfated K5 with high sulfation degree (K5-OS(H)) and N,O-sulfated K5 with high (K5-N,OS(H)) or low (K5-N,OS(L)) sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV-LTR-transactivation. Also, K5-OS(H) and K5-N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor-2 on endothelial cell (EC) surface. Finally, K5-OS(H) inhibits alphav beta3 integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5-OS(H) and K5-N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti-AIDS therapies.     

Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.     

Influence of diets supplemented with vitamins C and E on pirarucu (Arapaima gigas) blood parameters

This study evaluated the influence of diets supplemented with 500, 800, 1200 mg kg-1 of vitamin C (ascorbic acid or AA) and vitamin E (alpha-tocopherol or alpha-T) on the physiological responses of pirarucu fed for 2 months. Weight and mortality were not affected by dietary vitamin type or their concentrations. Significant increase (p<0.05) on the red blood cells count was obtained on treatments with 800 and 1200 mg AA kg-1 and on the hemoglobin concentration on treatment with 500 mg alpha-T kg-1 relatively to control. Mean corpuscular volume presented a significant decrease (p<0.05) on treatment with 800 and 1200 mg AA kg-1 when compared to control. Mean corpuscular hemoglobin concentration was significantly high (p<0.05) on treatment with 500 mg alpha-T kg-1. Only in vitamin C treatments, we noticed a significant increase (p<0.05) in the number of leucocytes relative to control. All fish in the vitamin-supplemented treatments, except 500 mg AA kg-1, had high total protein values compared to control. Fish treated with 800 or 1200 mg alpha-T kg-1 also showed increases in plasma glucose concentrations. Our results suggest that 800 and 1200 mg AA kg-1 are probably the most suitable concentrations for pirarucu diets, although high vitamin E diets are not necessary for quantitative leucocyte increases for this species.     

Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.     

Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of the family have all been implicated in membrane protein biogenesis of respiratory and energy transducing proteins. The number of proteins identified thus far to require YidC for their membrane biogenesis remains limited and the identification of new substrates may allow the elucidation of properties that define the YidC specificity. To this end we investigated changes in the membrane proteome of E. coli upon YidC depletion using metabolic labeling of proteins with ¹⁵N/¹⁴N combined with a MS‐centered proteomics approach and compared the effects of YidC depletion under aerobic and anaerobic growth conditions. We found that YidC depletion resulted in protein aggregation/misfolding in the cytoplasm as well as in the inner membrane of E. coli. A dramatic increase was observed in the chaperone‐mediated stress response upon YidC depletion and this response was limited to aerobically grown cells. A number of transporter proteins were identified as possible candidates for the YidC‐dependent insertion and/or folding pathway. These included the small metal ion transporter CorA, numerous ABC transporters, as well as the MFS transporters KgtP and ProP, providing a new subset of proteins potentially requiring YidC for membrane biogenesis.     

Structure of human chitotriosidase. Implications for specific inhibitor design and function of mammalian chitinase-like lectins

Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.     

SPO11-Independent DNA Repair Foci and Their Role in Meiotic Silencing

In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11^YF/YF), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11^YF/YF and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.     

Agonistic trials with mirrors do not elicit the same aggressiveness of a real trial in the matrinxã fish,Brycon amazonicus (Spix & Agassiz, 1829)

This study tested the efficacy of mirror trials in studying aggressiveness in the matrinxã fish, Brycon amazonicus. The hypothesis was that a mirror would elicit an aggressive response in B. amazonicus juveniles, but show different behavioral and physiological profiles than those observed in trials with real opponents. Fish were tested using either a mirror trial (n = 7) or a real trial (n = 7), that is, placed alone with a mirror or paired with a same‐sized opponent, respectively. All trials lasted for 20 min and took place in 96‐L aquaria with water temperatures of 28°C. Fish in mirror trials exhibited less locomotion (mirror: 423.3 ± 39.1; real trial: 735.1 ± 31.9; p < .001), rushed less against the opponent (mirror: 3.1 ± 2.3; real trial: 39.6 ± 5.1; p < .001) and showed less escalation of aggressive behavior compared to the winner of a real trial. Regarding the physiological parameters, despite similar cortisol responses (mirror: 84.2 ± 13.8 ng/ml; real trial: 77.2 ± 16.9 ng/ml; p = .757), the fish that fought against a mirror had lower levels of plasma glucose than those of fish that fought against a real opponent (mirror: 76.8 ± 3.6 ng/dl; real trial: 103.8 ± 10.1 ng/ml; p = .037). This lower energy mobilization might be due to a lower cost of the fight in the absence of an opponent that flees from attacks, as the locomotion and number of rush totals were also lower in the mirror trials. This pattern of less locomotion is probably common in mirror trials because the opponent is always limited to one side of the arena. If the energetic cost is one of the factors that can determine the outcome of a fight, and if mirror trials do not evoke the physiological response observed in a real fight, the use of mirror trials must be reviewed in studies that consider the physiological responses.     

The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins

Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.