Anaphylaxis induced by Thalassophryne nattereri venom in mice is an IgE/IgG1-mediated, IL-4-dependent phenomenon.

We hypothesized that beyond the Thalassophryne nattereri venoms ability to induce in mice a strong specific-Th2 response with high levels of specific IgE/IgG1, it would be able to trigger anaphylaxis in sensitized individuals. To investigate whether the venom is capable of inducing an allergic reaction in mice and characterize soluble and cellular mediators involved in this process, BALB/c female mice were sensitized intraperitoneally with decreasing-dose of venom at weekly intervals for 4 weeks and challenged by intraperitoneal, oral or epicutaneous routes with venom 2 weeks later. Our data show that sensitized-mice challenged by all routes showed intense symptoms of anaphylaxis, dependent on the anaphylactic IgG1 and IgE antibodies and mast cells. The late-phase reaction developed after initial symptoms was characterized by the influx of eosinophils, dependent on IL-5, IL-17A and eotaxin produced by Th2 cells in inflamed lungs and skin draining lymph-nodes. Using C57BL/6 deficient mice we demonstrated that IL-4 KO mice failed to develop anaphylactic symptoms or local Th2 inflammation, producing low levels of IgG1 and increased levels of IgG2a. Together our results demonstrated that the venom of T. nattereri has allergenic proteins that can trigger an allergic process, a phenomenon IgE-IgG1 dependent, IL-4-mediated and negatively regulated by IFN-γ.

Angiotensin converting enzyme of Thalassophryne nattereri venom.

Animal venoms are complex mixtures, including peptides, proteins (i.e., enzymes), and other compounds produced by animals in predation, digestion, and defense. These molecules have been investigated regarding their molecular mechanisms associated with physiological action and possible pharmacological applications. Recently, we have described the presence of a type of angiotensin converting enzyme (ACE) activity in the venom of Thalassophryne nattereri. It is a zinc-dependent peptidase with a wide range of effects. By removing dipeptide His-Leu from terminal C, the ACE converts angiotensinI (AngI) into angiotensin II (AngII) and inactivates bradykinin, there by regulating blood pressure and electrolyte homeostasis. The fractionation of T. nattereri venom in CM-Sepharose indicated a peak (CM2) with angiotensin-converting activity, converting AngI into Ang II. Electrophoresis on polyacrylamide gel (12%) revealed one band with 30kDa for CM2 similar in size to natterins, which are toxins with proteolytic activity found in T. nattereri venom. Mass spectrometry indicated that the protein sequence of the ACE purified from T. nattereri venom corresponds to natterin 1. The isolated protein has also demonstrated inhibition through captopril and EDTA and is characterized as a classic ACE. Thus, the isolated enzyme purified from T. nattereri venom is the first ACE isolated from fish venom.

Crude protein requeriment to pacamã juveniles / Exigência de proteína bruta para juvenis de pacamã

Pacamã (Lophiosilurus alexandri) is a species of carnivorous fish, suitable for farming, but little studied. The aim of this study was to determinate crude protein (CP) requirement to pacamã, L. alexandri, in juvenile phase, an experiment was conducted during 45 days in a completely randomized design, with five treatments (36.2, 38.2, 42.0, 44.4, and 48.8% CP of diet) and four replications, where a hundred juvenile fishes (5.19 ± 0.01 g) were distributed in twenty 36 L aquaria. Each aquarium with five juveniles was considered an experimental unit. The parameters evaluated were final weight, percentage weight gain, specific growth rate, survival rate, hepatosomatic index, carcass yield with and without head, total length, head length, width, and height. By the regression analysis was verify a (P0.05). Thus, it is recommended to use feed containing 36.2% of crude protein to pacamã (L. alexandri) juveniles.

O pacamã (Lophiosilurus alexandri) é uma espécie de peixe com hábito alimentar carnívoro, apropriada para o cultivo, porém, ainda pouco estudada. O objetivo deste estudo foi determinar a exigência de proteína bruta (PB) do pacamã, L. alexandri, na fase juvenil. O experimento foi conduzido por um período de 45 dias utilizando um delineamento inteiramente casualizado, com cinco tratamentos (dieta contendo 36,2; 38,2; 42,0; 44,4 e 48,8% de PB) e quatro repetições cada, onde 100 juvenis (5,19 ± 0,01g) foram distribuídos em 20 caixas com volume útil de 36 L, sendo considerada como unidade experimental uma caixa com cinco juvenis. Foram avaliados os parâmetros de peso final, percentagem de ganho de peso, taxa de crescimento específico, sobrevivência, índice hepatossomático, rendimento de carcaça com e sem cabeça, comprimento total, comprimento da cabeça, largura e altura. Pela análise de regressão foi verificada uma redução linear (P0,05). Deste modo, recomenda-se a utilização de rações contendo entre 36,2% de proteína bruta para juvenis de pacamã (L. alexandri).

Crude protein requeriment to pacamã juveniles / Exigência de proteína bruta para juvenis de pacamã

Pacamã (Lophiosilurus alexandri) is a species of carnivorous fish, suitable for farming, but little studied. The aim of this study was to determinate crude protein (CP) requirement to pacamã, L. alexandri, in juvenile phase, an experiment was conducted during 45 days in a completely randomized design, with five treatments (36.2, 38.2, 42.0, 44.4, and 48.8% CP of diet) and four replications, where a hundred juvenile fishes (5.19 ± 0.01 g) were distributed in twenty 36 L aquaria. Each aquarium with five juveniles was considered an experimental unit. The parameters evaluated were final weight, percentage weight gain, specific growth rate, survival rate, hepatosomatic index, carcass yield with and without head, total length, head length, width, and height. By the regression analysis was verify a (P<0.01) linear decrease to performance parameters in function to ration protein growth, however to Tukey test the best (P<0.01) result was obtained with 36.2% of CP. There was no influence of treatments on the other evaluated parameters (p>0.05). Thus, it is recommended to use feed containing 36.2% of crude protein to pacamã (L. alexandri) juveniles.(AU)

O pacamã (Lophiosilurus alexandri) é uma espécie de peixe com hábito alimentar carnívoro, apropriada para o cultivo, porém, ainda pouco estudada. O objetivo deste estudo foi determinar a exigência de proteína bruta (PB) do pacamã, L. alexandri, na fase juvenil. O experimento foi conduzido por um período de 45 dias utilizando um delineamento inteiramente casualizado, com cinco tratamentos (dieta contendo 36,2; 38,2; 42,0; 44,4 e 48,8% de PB) e quatro repetições cada, onde 100 juvenis (5,19 ± 0,01g) foram distribuídos em 20 caixas com volume útil de 36 L, sendo considerada como unidade experimental uma caixa com cinco juvenis. Foram avaliados os parâmetros de peso final, percentagem de ganho de peso, taxa de crescimento específico, sobrevivência, índice hepatossomático, rendimento de carcaça com e sem cabeça, comprimento total, comprimento da cabeça, largura e altura. Pela análise de regressão foi verificada uma redução linear (P<0.01) nos parâmetros de desempenho em função do nível protéico da ração. Quando comparados pelo teste de Tukey, o melhor resultado (P<0.01) foi obtido com 36,2% de PB. Não houve influência dos tratamentos nos outros parâmetros avaliados (p>0,05). Deste modo, recomenda-se a utilização de rações contendo entre 36,2% de proteína bruta para juvenis de pacamã (L. alexandri).(AU)

Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to extracellular matrix degradation.

Symptoms evoked by Thalassophryne nattereri fish envenomation include local oedema, severe pain and intense necrosis with strikingly inefficient healing, continuing for several weeks or months. Investigations carried out in our laboratory showed that, in the venom-induced acute inflammation, thrombosis in venules and constrictions in arterioles were highly visible, in contrast to a notable lack of inflammatory cell. Nevertheless, the reason that the venom toxins favour delayed local inflammatory response is poorly defined. In this study, we analysed the movement of leucocytes after T. nattereri venom injection in the intraplantar region of Swiss mice, the production of pro-inflammatory mediators and the venom potential to elicit matrix metalloproteinase production and extracellular matrix degradation. Total absence of mononuclear and neutrophil influx was observed until 14 days, but the venom stimulates pro-inflammatory mediator secretion. Matrix metalloproteinases (MMP)-2 and MMP-9 were detected in greater quantities, accompanied by tissue degradation of collagenous fibre. An influx of mononuclear cells was noted very late and at this time the levels of IL-6, IL-1beta and MMP-2 remained high. Additionally, the action of venom on the cytoskeletal organization was assessed in vitro. Swift F-actin disruption and subsequent loss of focal adhesion was noted. Collectively these findings show that the altered specific interaction cell-matrix during the inflammatory process creates an inadequate environment for infiltration of inflammatory cells.

Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom.

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.