Deficient skeletal muscle regeneration, which often leads to permanent sequelae, is a common clin... more Deficient skeletal muscle regeneration, which often leads to permanent sequelae, is a common clinical finding in envenomations caused by snakes of the family Viperidae, such as those of Bothrops alternatus and B. diporus in South America. The causes of such poor muscle regenerative outcome are still incompletely understood. Using a murine experimental model of envenomation by the venoms of these two species, we assessed whether traces of venom components that remain in muscle tissue days after envenomation affect myoblasts and myotube formation in culture. The kinetics of drop in venom concentration in the tissue was assessed by ELISA and Western blot, and by the quantification of venom phospholipase A2 activity. A rapid drop of venom components was observed in muscle, although a band of 58-63 kDa remained even 168 h after venom injection, and venom phospholipase A2 activity was detected in muscle tissue days after envenomation. Muscle homogenates from envenomated animals were cytotoxic to myoblasts in culture and inhibited the formation of myotubes even in conditions where homogenates were devoid of cytotoxicity. These deleterious effects were abrogated when homogenates were incubated with antivenom. Our findings agree with previous observations with the venom of Bothrops asper and provide further evidence that one of the causes of the poor skeletal muscle regeneration after Bothrops sp venom-induced myonecrosis is the deleterious action on myogenic cells of traces of venom components remaining in the tissue.
Thermal decomposition of 3,3,6,6-tetramethy l-1,2,4,5-tetroxane in presence of iron (III) salts t... more Thermal decomposition of 3,3,6,6-tetramethy l-1,2,4,5-tetroxane in presence of iron (III) salts takes place through a coordination mechanism, involving the metal ion as a Lewis acid, and it's completely consistent with the counter ion effect observed. The activation parameters of the reaction initial step (∆H # = 10,96±0.8 kcal/mol ; ∆S # = -34,4±0.6 kcal/mol; ∆G # = 25,48±0.8 kcal/mol) are compared with those reported for the unimolecular thermolyses of the 3,3,6,6- tetramethyl-1,2,4,5-tetroxane in presence of other metallic ions.
El objetivo del trabajo fue evaluar actividades farmacológicas (citotóxica, bactericida, agregan... more El objetivo del trabajo fue evaluar actividades farmacológicas (citotóxica, bactericida, agregante plaquetaria) y factores que afectan la actividad/ estabilidad de la fosfolipasa A2 (PLA2) aislada del veneno de Bothrops alternatus. Se utilizaron mioblastos/miotúbulos murinos (C2C12) y cepas bacterianas de Staphylococcus aureus (cepa ATCC 25923) y Escherichia coli (cepa ATCC 25922) para valorar respectivamente su acción miotóxica (in vitro) y bactericida, como así también líneas celulares epiteliales mamarias normales (NMuMG) y tumorales (LM3) para detectar su posible aplicación en terapia oncológica. Con el fin de evaluar la acción inhibitoria de la PLA2 aislada sobre la agregación plaquetaria se empleó plasma rico en plaquetas y trombina como inductor fisiológico. Por último, se registraron los cambios en la actividad y estabilidad de la PLA2 a diferentes temperaturas (4-90ºC) mediante ensayo cinético, en tanto que, por hemólisis radial indirecta se hizo lo mismo para evaluar e...
Con el objeto de avalar la sensibilidad del veneno ofídico sobre la densa red vascular que irriga... more Con el objeto de avalar la sensibilidad del veneno ofídico sobre la densa red vascular que irriga el pie equino, se analizaron los efectos in vitro de 50 μg de veneno, a través de biopsias de talones de 5 equinos de raza indefinida. Las muestras se incubaron en placas de cultivo celular conteniendo medio DMEM (Dulbecco´s Modified Eagle´s Medium). Un grupo de muestras operó como control y otro fue tratado con veneno, en estufa a 37ºC, con 5% de dióxido de carbono y 95% de humedad durante 24 y 48 h. Concluida la incubación, las muestras se fijaron en formaldehído bufferado y se procesaron con las técnicas clásicas para histopatología, siendo teñidas con HyE y PAS para su observación en microscopio óptico. Las muestras destinadas a microscopía electrónica de transmisión (MET) fueron tratadas por el método clásico. En los cortes coloreados (HyE) las arteriolas presentaron desprendimiento de células endoteliales, los núcleos fueron fuertemente basófilos, aumentados de tamaño y de forma r...
Bothrops diporus, previously considered a subspecies of the B. neuwiedi complex, is a medically r... more Bothrops diporus, previously considered a subspecies of the B. neuwiedi complex, is a medically relevant viperid in Northeastern Argentina. The venom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, and edema. In the present study, two basic phospholipases A2 (PLA2-I and PLA2-II) were isolated from this venom, and their pathological effects upon murine skeletal muscle and myogenic cells in culture were analyzed. Partial amino acid sequencing showed that PLA2-I and PLA2-II are Asp49 and Lys49 PLA2s, respectively. In agreement with this, PLA2-I showed PLA2 activity, whereas PLA2-II did not. Functional assays revealed differences in their myotoxicity, cytotoxicity, and anti-adhesion activity, and in the ability to inhibit cell migration, all of which were greater for the Lys49 variant. Native electrophoresis showed that PLA2-I was less basic than PLA2-II. The two proteins act synergistically to affect the integrity of C2C12 myogenic cells, providing a further example of the concerted action of coexisting snake venom components. PLA2-I and PLA2-II, together with additional basic PLA2s revealed by RP-HPLC, probably play an important role in myonecrosis after envenomation by B. diporus.
Acid proteases from sábalo stomach mucosa were recovered using salting-out procedure. This single... more Acid proteases from sábalo stomach mucosa were recovered using salting-out procedure. This single step produced an enzyme extract purified 1.8-fold over the crude extract with a recovery of 45.1% of its initial proteolytic activity. Sábalo proteases exhibited the highest activity at 45 °C-pH 2.0, showed pH stability between 2.0 and 5.0 and retained more than 70% of its activity after incubation at pH 7.0 for 2 h. Fish extract was unstable at temperatures greater than 45 °C. Its activity was inhibited by pepstatin A but not by PMSF, while EDTA and SDS showed partial inhibitory effects. Presence of CaCl and MgCl increased the proteolytic activity, while increasing concentrations of NaCl strongly decreased it. In addition, compared to the acid extraction method, the use of sábalo enzymatic extract increased 1.7 times the yield of collagen extraction.
Apoptosis : an international journal on programmed cell death, 2017
In this study, the apoptosis inducing effects of baltergin as well as its influence on cell adhes... more In this study, the apoptosis inducing effects of baltergin as well as its influence on cell adhesion and migration on muscles cells in vitro were studied. Morphological analysis made by scanning electron and phase contrast microscopy demonstrated typical futures of programmed cell death, apoptosis. This mechanism was confirmed by fluorescence staining, molecular analysis of endonuclease activity and increased mRNA expression level of two representative genes (p53 and bax). On the other hand, baltergin exert an inhibition effect on myoblast cell adhesion and migration in vitro probably through a mechanism that involves the interaction of this enzyme with cell integrins. In conclusion, our results suggest that the absence of appropriate extracellular matrix contacts triggers anoikis. Therefore, this is the first report that demonstrated the mechanism of programmed cell death triggered by baltergin, a PIII metalloprotease isolated from Bothrops alternatus venom, in a myoblast cell line.
Deficient skeletal muscle regeneration, which often leads to permanent sequelae, is a common clin... more Deficient skeletal muscle regeneration, which often leads to permanent sequelae, is a common clinical finding in envenomations caused by snakes of the family Viperidae, such as those of Bothrops alternatus and B. diporus in South America. The causes of such poor muscle regenerative outcome are still incompletely understood. Using a murine experimental model of envenomation by the venoms of these two species, we assessed whether traces of venom components that remain in muscle tissue days after envenomation affect myoblasts and myotube formation in culture. The kinetics of drop in venom concentration in the tissue was assessed by ELISA and Western blot, and by the quantification of venom phospholipase A2 activity. A rapid drop of venom components was observed in muscle, although a band of 58-63 kDa remained even 168 h after venom injection, and venom phospholipase A2 activity was detected in muscle tissue days after envenomation. Muscle homogenates from envenomated animals were cytotoxic to myoblasts in culture and inhibited the formation of myotubes even in conditions where homogenates were devoid of cytotoxicity. These deleterious effects were abrogated when homogenates were incubated with antivenom. Our findings agree with previous observations with the venom of Bothrops asper and provide further evidence that one of the causes of the poor skeletal muscle regeneration after Bothrops sp venom-induced myonecrosis is the deleterious action on myogenic cells of traces of venom components remaining in the tissue.
Thermal decomposition of 3,3,6,6-tetramethy l-1,2,4,5-tetroxane in presence of iron (III) salts t... more Thermal decomposition of 3,3,6,6-tetramethy l-1,2,4,5-tetroxane in presence of iron (III) salts takes place through a coordination mechanism, involving the metal ion as a Lewis acid, and it's completely consistent with the counter ion effect observed. The activation parameters of the reaction initial step (∆H # = 10,96±0.8 kcal/mol ; ∆S # = -34,4±0.6 kcal/mol; ∆G # = 25,48±0.8 kcal/mol) are compared with those reported for the unimolecular thermolyses of the 3,3,6,6- tetramethyl-1,2,4,5-tetroxane in presence of other metallic ions.
El objetivo del trabajo fue evaluar actividades farmacológicas (citotóxica, bactericida, agregan... more El objetivo del trabajo fue evaluar actividades farmacológicas (citotóxica, bactericida, agregante plaquetaria) y factores que afectan la actividad/ estabilidad de la fosfolipasa A2 (PLA2) aislada del veneno de Bothrops alternatus. Se utilizaron mioblastos/miotúbulos murinos (C2C12) y cepas bacterianas de Staphylococcus aureus (cepa ATCC 25923) y Escherichia coli (cepa ATCC 25922) para valorar respectivamente su acción miotóxica (in vitro) y bactericida, como así también líneas celulares epiteliales mamarias normales (NMuMG) y tumorales (LM3) para detectar su posible aplicación en terapia oncológica. Con el fin de evaluar la acción inhibitoria de la PLA2 aislada sobre la agregación plaquetaria se empleó plasma rico en plaquetas y trombina como inductor fisiológico. Por último, se registraron los cambios en la actividad y estabilidad de la PLA2 a diferentes temperaturas (4-90ºC) mediante ensayo cinético, en tanto que, por hemólisis radial indirecta se hizo lo mismo para evaluar e...
Con el objeto de avalar la sensibilidad del veneno ofídico sobre la densa red vascular que irriga... more Con el objeto de avalar la sensibilidad del veneno ofídico sobre la densa red vascular que irriga el pie equino, se analizaron los efectos in vitro de 50 μg de veneno, a través de biopsias de talones de 5 equinos de raza indefinida. Las muestras se incubaron en placas de cultivo celular conteniendo medio DMEM (Dulbecco´s Modified Eagle´s Medium). Un grupo de muestras operó como control y otro fue tratado con veneno, en estufa a 37ºC, con 5% de dióxido de carbono y 95% de humedad durante 24 y 48 h. Concluida la incubación, las muestras se fijaron en formaldehído bufferado y se procesaron con las técnicas clásicas para histopatología, siendo teñidas con HyE y PAS para su observación en microscopio óptico. Las muestras destinadas a microscopía electrónica de transmisión (MET) fueron tratadas por el método clásico. En los cortes coloreados (HyE) las arteriolas presentaron desprendimiento de células endoteliales, los núcleos fueron fuertemente basófilos, aumentados de tamaño y de forma r...
Bothrops diporus, previously considered a subspecies of the B. neuwiedi complex, is a medically r... more Bothrops diporus, previously considered a subspecies of the B. neuwiedi complex, is a medically relevant viperid in Northeastern Argentina. The venom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, and edema. In the present study, two basic phospholipases A2 (PLA2-I and PLA2-II) were isolated from this venom, and their pathological effects upon murine skeletal muscle and myogenic cells in culture were analyzed. Partial amino acid sequencing showed that PLA2-I and PLA2-II are Asp49 and Lys49 PLA2s, respectively. In agreement with this, PLA2-I showed PLA2 activity, whereas PLA2-II did not. Functional assays revealed differences in their myotoxicity, cytotoxicity, and anti-adhesion activity, and in the ability to inhibit cell migration, all of which were greater for the Lys49 variant. Native electrophoresis showed that PLA2-I was less basic than PLA2-II. The two proteins act synergistically to affect the integrity of C2C12 myogenic cells, providing a further example of the concerted action of coexisting snake venom components. PLA2-I and PLA2-II, together with additional basic PLA2s revealed by RP-HPLC, probably play an important role in myonecrosis after envenomation by B. diporus.
Acid proteases from sábalo stomach mucosa were recovered using salting-out procedure. This single... more Acid proteases from sábalo stomach mucosa were recovered using salting-out procedure. This single step produced an enzyme extract purified 1.8-fold over the crude extract with a recovery of 45.1% of its initial proteolytic activity. Sábalo proteases exhibited the highest activity at 45 °C-pH 2.0, showed pH stability between 2.0 and 5.0 and retained more than 70% of its activity after incubation at pH 7.0 for 2 h. Fish extract was unstable at temperatures greater than 45 °C. Its activity was inhibited by pepstatin A but not by PMSF, while EDTA and SDS showed partial inhibitory effects. Presence of CaCl and MgCl increased the proteolytic activity, while increasing concentrations of NaCl strongly decreased it. In addition, compared to the acid extraction method, the use of sábalo enzymatic extract increased 1.7 times the yield of collagen extraction.
Apoptosis : an international journal on programmed cell death, 2017
In this study, the apoptosis inducing effects of baltergin as well as its influence on cell adhes... more In this study, the apoptosis inducing effects of baltergin as well as its influence on cell adhesion and migration on muscles cells in vitro were studied. Morphological analysis made by scanning electron and phase contrast microscopy demonstrated typical futures of programmed cell death, apoptosis. This mechanism was confirmed by fluorescence staining, molecular analysis of endonuclease activity and increased mRNA expression level of two representative genes (p53 and bax). On the other hand, baltergin exert an inhibition effect on myoblast cell adhesion and migration in vitro probably through a mechanism that involves the interaction of this enzyme with cell integrins. In conclusion, our results suggest that the absence of appropriate extracellular matrix contacts triggers anoikis. Therefore, this is the first report that demonstrated the mechanism of programmed cell death triggered by baltergin, a PIII metalloprotease isolated from Bothrops alternatus venom, in a myoblast cell line.
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