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A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56μg/mL) and Trypanosoma cruzi (IC50=4.85μg/mL). This toxin also induced cytotoxicity (IC50=1.80μg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

Background: L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. Methods and results: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4°C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. Conclusions: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.

Cryoglobulins are cold-precipitable immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders. Their appearance along with rheumatoid factor (RF) can be considered a normal event in the clearance of immune complexes and rarely produces any symptoms. The association between hepatitis C virus (HCV) and mixed cryoglobulinemia (MC) has been rendered evident since the recognition of serological markers of HCV infection. There is thus every reason to suppose that direct or indirect involvement of B cells on the part of the HCV results in their persistent stimulation, clonal expansion and release of molecules with RF activity. The formation of RF/IgG immune complexes is the key pathogenetic mechanism. The close correlation between HCV infection and MC also throws new light on the interpretation of autoimmune phenomena in the course of viral infection and on the close link between autoimmune diseases and lymphoproliferative disorders. The higher risk of non-Hodgkin's lymphoma (NHL) displayed by HCV positive subjects, especially in the Mediterranean basin, suggests that the HCV's chronic lymphoproliferative drive may progress towards frank lymphoid neoplasia. The presence of MC does not represent an in situ or 'occult' NHL, because recent evidences indicate that none of the clones interpreted as predominant displays the molecular features of a true neoplastic process. The cryoglobulinemic syndrome is probably the consequence of pathogenic noxae that act upon the immune system of a host in which regulation of the peripheral T cell response appears to be in some way altered.

The synthesis and spectroscopic characterization of self-assembled cylindrical capsule 1a x 1a of nanometer dimensions is described. Encapsulation studies of large organic guest molecules were performed by using 1H NMR sprectroscopy in [D12]mesitylene solution. In addition to the computational (MacroModel 5.5, Amber* force field) analysis of the capsule's shape and geometry, an experimental approach towards estimation of the internal cavity dimensions is described. This involves using series of homologous molecular "rulers" (e.g. aromatic amides 5a-i). The available space inside the capsule 1a x 1a can be estimated as 5.7 x 14.7 A (error +/- 0.2 A) with this technique. Dibenzoyl peroxide is readily encapsulated in [D12]mesitylene and was shown to be stable to decomposition for at least three days at 70 degrees C inside the capsule. Moreover, 1a x 1a prevents the encapsulated peroxide from oxidizing Ph3P or diphenyl carbazide present in solution. The normal chemical reactivity of the peroxide is restored by release from the capsule by DMF, a solvent that competes for the hydrogen bonds that hold the capsule together. The protection and release of encapsulated species augurs well for the application of capsules in catalysis and delivery.

The loss of hearing abilities can be seen as a complex event because psychological discomfort and relational inhibitions interact and lead to behaviours and attitudes which are insidious and dangerous for the quality of life of the patient. The complexity of the psychological-organic inconvenience, such as impairement disability and handicap led to the testing of recovery patterns. In this specific context interventions coming from different fields sustained from a complex operative model where different professional competences and evaluation processes interact. Therapeutic interventions have been carried out by an audiologist, an audiometrist, a speech therapist, an audioprostethist and a psychologist, all sharing the obtaining of the same result. The role of the audiologist is simply clinical and concerns the knowledge of the entire process of rehabilitation through the use of a prosthesis. This process concerns the audiologist, who operates in close collaboration with the audiometrist and the audioprosthesist: it is therefore a therapeutic activity that is interested mainly in the prescription of the prosthesis and the restoring of the communicative function. The speech therapist and the psychologist carry their interventions through a relationship with the patient, which places the respect of the patient's personality before any other procedural and technical aspect. Therefore they pay attention more to ergonomic factors than to the hearing loss, through the obtaining of the patient's self-confidence and of a better general psychological situation. Therefore the main purpose of each intervention is to create a process of rehabilitation aimed at restoring the communicative functions and the individual motivation in trying to do so both in the domestic and in the social environment. The authors refer the experience and the informations put together during three years of research activity. The results of the therapeutic intervention have brought to the acceptance of the prosthesis help, the adaptation to amplification, the reduction of the subjective and relational uneasiness for the use of prosthesis, the use of the prosthesis, the reintroduction to the world of sounds, the restoring of levels of autonomy and of self-estime, the discover of eventual abilities, which had never been used or underestimated, the reactivation of more rewarding social relationships and the reduction of the conditions of dependency related to the hearing disability.