Papers by Soledad Bustillo
Journal of Ethnopharmacology
Proceedings of 1st International Electronic Conference on Toxins, 2021
Toxicon, 2022
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.
Toxicon, 2020
The prolonged consumption of Ipomoea carnea produces neurologic symptoms in animals and a typical... more The prolonged consumption of Ipomoea carnea produces neurologic symptoms in animals and a typical histological lesion, cytoplasmic vacuolization, especially in neurons. The toxic principles of I. carnea are the alkaloids swainsonine and calystegines B 1 , B 2 , B 3 and C 1. In this study, primary brain cultures from newborn mouse containing mixed glial cells were utilized. These cells were exposed to Ipomoea extracts containing between 0 and 250 μM swainsonine for 48 h. Morphological changes were investigated through Phase Contrast microscopy and Rosenfeld's staining. The extract induced cytoplasmic vacuolization in astrocytes and microglia in a dose dependent manner, being more evident when cultures were exposed to 250 μM of swainsonine. In addition, acridine orange staining evidenced an increase in the number of lysosomes in both microglia and astrocytes cells. Consistent with this, scanning electron microscopy also showed that both types of cells presented morphological characteristics of cell activation. Ultrastructurally, cells showed vacuoles filled with amorphous material and surrounded by a single membrane and also multilayer membranes. Taken together, these findings suggest that swainsonine along with calystegines, are probably responsible for the activation of glial cells due to a possible lysosomal dysfunction and therefore intracellular storage. Our results demonstrate that this in vitro glial cell model is a very good alternative to in vivo studies that require several weeks of animal intoxication to observe similar neurotoxic effects.
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.
This study was undertaken to evaluate the antimicrobial activity on Gram-negative and Gram-positi... more This study was undertaken to evaluate the antimicrobial activity on Gram-negative and Gram-positive bacteria of the venom from Bothrops alternatus, specie responsible for most of snakebites in Northeastern Argentina. This activity was investigated against Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Enterococcus faecalis (ATCC 29212). Inhibitory halos values and reduction of colony-forming unites (CFU) by dilution plate technique were determined. The bactericidal activity was higher for E. coli and S. aureus, in comparison to that for P. aeruginosa. E. faecalis was the most resistant organism tested to the action of venom solutions. The demonstrated bactericidal property of B. alternatus venom highlight it as a promising candidate for further studies according to detect which component, or components, are responsible of this activity, in order to then investigate their potential use as antimicrobial agent.
Toxicon : official journal of the International Society on Toxinology, 2020
Europe is inhabited by medically important venomous Viperinae snakes. Vipera ammodytes, Vipera be... more Europe is inhabited by medically important venomous Viperinae snakes. Vipera ammodytes, Vipera berus and Vipera aspis cause the greatest public health problem in Europe, but there are other equally significant snakes in specific regions of the continent. For example, Macrovipera lebetina and Montivipera xanthina are the most dangerous species in Turkey. Immunotherapy is indicated for patients with systemic envenoming, of which there are approximately 4,000 annual cases in Europe. In the present study, safety and venom-neutralizing efficacy of Inoserp™ Europe, a new F(ab') 2 polyvalent antivenom designed to cover envenoming by snakes of the European region, were evaluated. Horse hyperimmune plasmas were produced by immunization with the venoms of the following species: Vipera ammodytes, Vipera aspis, Vipera berus, Vipera latastei, Montivipera xanthina, Macrovipera schweizeri, Macrovipera lebetina obtusa, Macrovipera lebetina cernovi, and Macrovipera lebetina turanica. F(ab') 2 immunoglobulin fragments were obtained by enzymatic digestion with pepsin and precipitation with ammonium sulfate. In accordance with WHO recommendations, several quality control parameters were applied to evaluate the safety of this antivenom and the results showed that it meets those quality requirements such as the low protein content (10 g/dL) and the high level of immunoglobulin fragments F(ab') 2 (˃90%). The venom-neutralizing efficacy of the antivenom was evaluated in mice and the results showed that it had appropriate neutralizing potencies against the venoms of several species of the subfamily Viperinae; including V. ammodytes, V. berus, V. aspis, M. lebetina and M. xanthina in compliance with the European Pharmacopoeia. Paraspecificity of the antivenom was demonstrated as well, since it neutralized venoms of species not included in the immunization schemes. According to this assessment, Inoserp™ Europe satisfactorily meets the key WHO criteria from a quality standpoint. This is the first antivenom covering all WHO medical important species from western Europe to Caucasus region.
Toxicon, 2020
not hemorrhagic venom, but it is greater than that was reported for C. d.terrificus venom from Br... more not hemorrhagic venom, but it is greater than that was reported for C. d.terrificus venom from Brazil (0.96). The major LNC value it would be related to the low content ratio of crotamine (8%) in C.d.terrificus venom from Argentina. These results provide information about C.d.terrificus venom composition from Argentina and they could be considered on defining the mixture of venoms for immunization to produce an effective pan-American anti-Crotalus antivenom.
Toxicon, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revista Veterinaria, 2012
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...
Journal of Venomous Animals and Toxins including Tropical Diseases, 2020
Background: Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog speci... more Background: Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A 2 , clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 kDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A 2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.
Current Topics in Medicinal Chemistry, 2019
International Journal of Biological Macromolecules, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revista Veterinaria, 2017
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...
Toxicon, 2019
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.
Food chemistry, Jan 15, 2018
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.
International journal of biological macromolecules, Jan 19, 2018
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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Papers by Soledad Bustillo