Heloisa S. Selistre-de-Araujo

Androgenic-Anabolic Steroids Associated with Mechanical Loading Inhibit Matrix Metallopeptidase Activity and Affect the Remodeling of the Achilles Tendon in Rats:

Background The indiscriminate use of anabolic-androgenic steroids has been shown to induce pathologic changes in the Achilles tendon in several situations. Purpose To study tendon remodeling in rats treated with anabolic-androgenic steroids combined with an exercise program. Study Design Controlled laboratory study. Methods Wistar rats were grouped as follows: sedentary (group I), injected with anabolic-androgenic steroids only (group II), trained only (group III), and trained and injected with anabolic-androgenic steroids (group IV). The trained groups performed jumps in water: 4 series of 10 jumps each, with an overload of 50% to 70% of the animals body weight and a 30-second rest interval between series, for 6 weeks. Anabolic-androgenic steroids (5 mg/kg) were injected subcutaneously. Activity of matrix metallopeptidases, a marker for tendon remodeling, was analyzed in tissue extracts by zymography on gelatin–sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Results Morphological analyses of tendons showed that in group II, the most external layer that covers the tendon was thicker with aggregation of the collagen fibers, suggesting an increase in collagen synthesis. In group IV, an inflammatory infiltrate and fibrosis in tendons as well as a pronounced increase of the serum corticosterone level were observed. This training protocol upregulated matrix metallopeptidase activity, whereas anabolic-androgenic steroid treatment strongly inhibited this activity. The appearance of lytic bands with molecular masses of approximately 62 and 58 kDa suggests the activation of matrix metallopeptidase–2. Conclusion Anabolic-androgenic steroid treatment can impair tissue remodeling in the tendons of animals undergoing physical exercise by downregulating matrix metallopeptidase activity, thus increasing the potential for tendon injury. Clinical Relevance Since the AAS abuse is so widespread, a better comprehension of the pathological effects induced by these drugs may be helpful for the development of new forms of therapy of AAS-induced lesions.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys189 is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from PII SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu276 of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other PIII SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other PIII members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M(r)=34,000 under reducing conditions and pI approximately 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (-70 to 37 degrees C), pH values (3-9) or in the presence of divalent metal ions (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.

Snake Venom Disintegrins and Cell Migration

Cell migration is a key process for the defense of pluricellular organisms against pathogens, and it involves a set of surface receptors acting in an ordered fashion to contribute directionality to the movement. Among these receptors are the integrins, which connect the cell cytoskeleton to the extracellular matrix components, thus playing a central role in cell migration. Integrin clustering at focal adhesions drives actin polymerization along the cell leading edge, resulting in polarity of cell movement. Therefore, small integrin-binding proteins such as the snake venom disintegrins that inhibit integrin-mediated cell adhesion are expected to inhibit cell migration. Here we review the current knowledge on disintegrin and disintegrin-like protein effects on cell migration and their potential use as pharmacological tools in anti-inflammatory therapy as well as in inhibition of metastatic invasion.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2beta1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of approximately 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2beta1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2beta1 integrin.

Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K(v) channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na(V) channels (Na(V)1.4; Na(V)1.5; Na(V)1.6; Na(V)1.8 and DmNa(V)1) and 12 different subtypes of K(V) channels (K(V)1.1 - K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K(V)1.2 and K(V)1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na(V) channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.

Effects of resistance training on matrix metalloproteinase‐2 activity and biomechanical and physical properties of bone in ovariectomized and intact rats

The purpose of this study was to investigate the influence of resistance training on the activity of matrix metalloproteinase (MMP)‐2 and bone biomechanical properties in ovariectomized and intact rats. Forty‐eight female rats were divided into two distinct groups, ovariectomized (OVX) and intact (Int), which were subdivided into three similar subgroups: sedentary, acute exercise and chronic exercise. Rats performed a resistance training for 12 weeks in which animals climbed a vertical ladder of 1.1 m with weights attached to their tails. Sessions were performed with an interval of 3, 4–9 and 8–12 days scaled dynamic movements of climbing. Biomechanical and physical analyses were performed using a universal testing machine, and MMP‐2 activity analysis by zymography. Bone density (BD), mineral density (MD), maximum load and fracture load was reduced in sedentary and acute exercise OVX groups compared with the sedentary intact group (P<0.05); in contrast, chronically trained groups (OVX and Int) showed a significant increase in BD, MD and fracture load compared with all the other groups. MMP‐2 activity in chronically trained groups also showed a significant increase, while the sedentary OVX group showed a decrease in MMP‐2 activity compared with the intact sedentary group (P<0.05). Our results suggest that the resistance training proposed in our work was efficient in reverting the deleterious effects of ovariectomy on bone tissue, and also produced modeling effects in intact rats. On the other hand, ovariectomy reduced the activity of MMP‐2 and produced deleterious effects on bone tissue, mimicking menopause intrinsically.

Synthesis, characterization and cytotoxic activities of the [RuCl2(NO)(dppp)(L)]PF6 complexes

The synthesis and characterization of ruthenium compounds of the type [RuCl(2)(NO)(dppp)(L)]PF(6) [dppp=1,3-bis(diphenylphosphino)propane; L=pyridine, 4-methylpyridine, 4-phenylpyridine and dimethyl sulfoxide] are described. The complexes were characterized by elemental analysis, UV/Vis and infrared spectroscopy, cyclic voltammetry, and X-ray crystallography for the complexes with the pyridine and 4-methylpyridine ligands. In vitro evaluation of these nitrosyl complexes revealed cytotoxic activity from 7.1 to 19.0 microM against the MDA-MB-231 breast tumor cells and showed that, in this case, they are more active than the reference metallodrug cisplatin. The 1,3-bis(diphenylphosphino)propane and the N-heterocyclic ligands alone failed to show cytotoxic activities at the concentrations tested (maximum concentration utilized=200 microM).

Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the beta1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that alphaVbeta3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.