Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Neospora caninum: functional inhibition of protein disulfide isomerase by the broad-spectrum anti-parasitic drug nitazoxanide and other thiazolides

Nitazoxanide (NTZ) and several NTZ-derivatives (thiazolides) have been shown to exhibit considerable anti-Neospora caninum tachyzoite activity in vitro. We coupled tizoxanide (TIZ), the deacetylated metabolite, to epoxy-agarose-resin and performed affinity chromatography with N. caninum tachyzoite extracts. Two main protein bands of 52 and 43kDa were isolated. The 52kDa protein was readily recognized by antibodies directed against NcPDI, and mass spectrometry confirmed its identity. Poly-histidine-tagged NcPDI-cDNA was expressed in Escherichia coli and recombinant NcPDI (recNcPDI) was purified by Co2+-affinity chromatography. By applying an enzyme assay based on the measurement of insulin crosslinking activity, recNcPDI exhibited properties reminiscent for PDIs, and its activity was impaired upon the addition of classical PDI inhibitors such as bacitracin (1-2mM), para-chloromercuribenzoic acid (0.1-1mM) and tocinoic acid (0.1-1mM). RecNcPDI-mediated insulin crosslinking was inhibited by NTZ (5-100 microM) in a dose-dependent manner. In addition, the enzymatic activity of recNcPDI was inhibited by those thiazolides that also affected parasite proliferation. Thus, thiazolides readily interfere with NcPDI, and possibly also with PDIs from other microorganisms susceptible to thiazolides.

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

Inhibition of Wound-Induced Accumulation of Allene Oxide Synthase Transcripts in Flax Leaves by Aspirin and Salicylic Acid1

Allene oxide synthase (AOS) mediates the conversion of lipoxygenase-derived fatty acid hydroperoxides to unstable allene epoxides, which supply the precursors for the synthesis of the phytohormone jasmonic acid (JA). In this study the characterization of AOS gene expression in flax (Linum usitatissimum) is reported. AOS was constitutively expressed in different organs of flax plants. Additionally, AOS gene expression was enhanced after mechanical wounding in both the directly damaged leaves and in the systemic tissue located distal to the treated leaves. This wound-induced accumulation of AOS required the de novo biosynthesis of other unknown proteins involved in the signaling pathway modulating wound-induced AOS gene expression. Furthermore, the wound-induced AOS mRNA accumulation was correlated with the increase in the levels of JA. Both JA and its precursor, 12-oxo-phytodienoic acid, activated AOS gene expression in a dose-dependent manner. Thus, JA could activate its own biosynthetic pathway in flax leaves. Moreover, neither salicylic acid (SA) nor aspirin influenced AOS enzymatic activity. It is interesting that pretreatment with SA or aspirin inhibited wound-induced accumulation of AOS transcripts. These results suggest that a potent inhibition of JA biosynthetic capacity in leaves can be affected by SA or aspirin at the level of AOS gene expression.

Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Peptide conjugation to an in vitro-selected DNA ligand improves enzyme inhibition.

An in vitro selection technique was used to identify a specific high-affinity DNA ligand targeted to human neutrophil elastase (HNE). 1H NMR data and a comparative analysis of the selected sequences suggest that the DNA folds into a G-quartet structure with duplexed ends. The high-affinity binding DNA alone did not inhibit the enzymatic activity of HNE. The DNA was covalently attached to a tetrapeptide, N-methoxysuccinyl-Ala-Ala-Pro-Val, that is a weak competitive inhibitor of HNE. HNE was inhibited by this DNA-peptide conjugate nearly five orders of magnitude more effectively than by the peptide alone. These results demonstrate that in vitro-selected nucleic acids can be used as a vehicle for molecular delivery.

Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting and enzymatic assays as one of the 14-3-3-binding proteins in resting or activated cells. Moreover, endogenous 14-3-3 and PI3-K were coimmunoprecipitated from intact T cells. Far-Western blots of gel-purified, immunoprecipitated PI3-K with a recombinant 14-3-3 fusion protein revealed direct binding of 14-3-3 to the catalytic subunit (p110) of PI3-K. Finally, anti-phosphotyrosine immunoprecipitates from activated, 14-3-3-overexpressing cells contained lower PI3-K enzymatic activity than similar immunoprecipitates from control cells. These findings suggest that association of 14-3-3 with PI3-K in hematopoietic (and possibly other) cells regulates the enzymatic activity of PI3-K during receptor-initiated signal transduction.

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G(4)SG(4)) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 M, 30% glycerol, 1 mM CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR- H, IC50 &SIM; 1 μM). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

Inhibition of cell-wall autolysis and pectin degradation by cations

Modification of cell wall components such as cellulose, hemicellulose and pectin plays an important role in cell expansion. Cell expansion is known to be diminished by cations but it is unknown if this results from cations reacting with pectin or other cell wall components. Autolysis of cell wall material purified from bean root (Phaseolus vulgaris L.) occurred optimally at pH 5.0 and released mainly neutral sugars but very little uronic acid. Autolytic release of neutral sugars and uronic acid was decreased when cell wall material was loaded with Ca, Cu, Sr, Zn, Al or La cations. Results were also extended to a metal-pectate model system, which behaved similarly to cell walls and these cations also inhibited the enzymatic degradation by added polygalacturonase (EC 3.2.1.15). The extent of sugar release from cation-loaded cell wall material and pectate gels was related to the degree of cation saturation of the substrate, but not to the type of cation. The binding strength of the cations was assessed by their influence on the buffer capacity of the cell wall and pectate. The strongly bound cations (Cu, Al or La) resulted in higher cation saturation of the substrate and decreased enzymatic degradability than the weakly held cations (Ca, Sr and Zn). The results indicate that the junction zones between pectin molecules can peel open with weakly held cations, allowing polygalacturonase to cleave the hairy region of pectin, while strongly bound cations or high concentrations of cations force the junction zone closed, minimising enzymatic attack on the pectin backbone. (C) 2004 Elsevier SAS. All rights reserved.

Enzymatic stabilization of gelatin-based scaffolds

The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.

Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase

Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

Effects of Low Molecular Weight Sulfated Galactan Fragments From Botryocladia Occidentalis on the Pharmacological and Enzymatic Activity of Spla2 From Crotalus Durissus Cascavella

Low molecular weight fragments of sulfated galactans (Boc-5 and Boc-10) from the red algae Botryocladia occidentalis significantly inhibited Crotalus durissus cascavella sPLA2 enzymatic activity. Equimolar ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric inhibition of sPLA2. Under the conditions tested, we observed that both Boc-5 and Boc-10 strongly decreased edema, myonecrosis, and neurotoxicity induced by native sPLA2.

Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom

Flavonoids, coumarins and other polyphenolic compounds are powerful antioxiants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. Despite being widely used as powerful therapeutic agents for blood coagulation disorders, more specifically to control some serine protease enzymes, the mechanism of anti-inflammatory activity of coumarins is unknown, unlike that of flavonoids. Although their controlling effect on serine proteases is well acknowledged, their action on secretory phospholipase A2 (sPLA2) remains obscure. The present study describes the interaction between umbelliferone (7-HOC) and the sPLA2 from Crotalus durissus collilineatus venom. In vitro inhibition of sPLA2 enzymatic activity by 7-HOC was estimated using 4N3OBA as substrate, resulting in an irreversible decrease in such activity proportional to 7-HOC concentration. The biophysical interaction between 7-HOC and sPLA2 was examined by fluorescent spectral analysis and circular dichroism studies. Results from both techniques clearly showed that 7-HOC strongly modified the secondary structure of this enzyme and CD spectra revealed that it strongly decreased sPLA2 alphahelical conformation. In addition, two-dimensional electrophoresis indicated an evident difference between HPLC-purified native and 7-HOC-treated sPLA2s, which were used in pharmacological experiments to compare their biological activities. In vivo anti-inflammatory activity was assessed by the sPLA2-induced mouse paw edema model, in which 7-HOC presented an effect similar to those of dexamethasone and cyproheptacline against the pro-inflammatory effect induced by native sPLA2 on the mouse paw edema, mast cell degranulation and skin edema. on the other hand, 7-HOC exhibited a more potent inhibitory effect on sPUL2 than that of p-bromophenacyl bromide (p-BPB). Our data suggest that 7-HOC interacts with sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this enzyme, indicating its potential use to suppress inflammation induced by sPLA2 from the snake venom. (C) 2008 Published by Elsevier Ltd.

Ikkε Is Key To Induction Of Insulin Resistance In The Hypothalamus, And Its Inhibition Reverses Obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.