Luis Fernando Plenge-Tellechea

Snakebite envenomation is a public health problem in many areas in the world and is a significant cause of disability and death. Crotalid venoms consist of a cocktail of peptides and enzymes that can cause myonecrotoxic lesions, which are associated with irreversible loss of muscle tissue. The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) is a transmembrane protein with a critical role in maintaining cellular Ca²⁺ homeostasis, which is central in facilitating skeletal and cardiac muscle contraction/relaxation. Crotalid venom-induced myotoxicity has been linked to alterations in the intracellular levels of Ca²⁺. However, the specific mechanisms, including SERCA's involvement, are poorly understood. Thus, we investigated the in vitro toxic effect of crotalid venom on the enzymatic activity of SERCA, using venom of the Mexican black-tailed rattlesnake, Crotalus molossus nigrescens, (vCmn), and SERCA-enriched sarcoplasmic reticulum (SR) microsomes from rabbit skeletal muscle as experimental models. Enzymatic assays revealed significant vCmn-induced decreases in SERCA activity in a time- and dose-dependent manner. Thin layer chromatography and phospholipid hydrolysis measurements showed significant SR membrane damage. The results suggest that vCmn affects SERCA functionality and compromises the integrity of the SR membrane, both of which are critical for skeletal muscle function and could thus be key mediators of vCmn-induced myotoxicity.

The venom of Crotalus ornatus (vCo) poses a threat to human health, as it contains a mixture of toxins that can cause cytotoxic, necrotic, and hemolytic effects. The present study assessed methanolic and acetone extracts from leaves and flowers of Larrea tridentata, as well as the bark of Quercus virginiana as potential suppressors of the toxic effects of vCo in vitro. The content of total phenols, flavonoids, and tannins of the plant extracts were quantified for the suppression of vCo cytotoxicity in two cell culture models, human lymphocytes and porcine aortic endothelial (PAE) cells. Extracts from Q. virginiana displayed a greater concentration of total phenols, flavonoids, and tannins. Co-incubation of lymphocytes and PAE cells with fixed concentrations of vCo and plant extracts resulted in decreased vCo-induced cytotoxicity. A 24-hour co-incubation of lymphocytes with vCo (2.36 ± 0.17 µg/mL) and 0.5 µg/mL of methanolic leaf extract from L. tridentata (LLM) significantly suppressed the venom-induced cytotoxicity by 37.33 ± 8.33%. Similarly, the LLM extract (4 µg/mL) caused a significant decrease in vCo cytotoxicity after 24 hours in PAE cells. In contrast, while the acetone extract of Q. virginiana bark (QA) suppressed cytotoxicity by 29.20 ± 3.51% (p < 0.001) in lymphocytes, it failed to protect PAE cells against vCo after 24 hours. In PAE cells, a shorter 4-hour co-incubation showed significant suppression of cytotoxicity with both extracts. Our results collectively suggest that LLM and QA possess cytoprotective properties against the in vitro toxic effects of vCo, and thus establish extracts from these plants as potential therapeutic interventions against Crotalus envenomation.

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

Loxocelism is a neglected medical problem that depends on its severity, can cause a cutaneous or viscero-cutaneous syndrome. This syndrome is characterized by hemostatic effects and necrosis, and the severity of the loxoscelism depends on the amount of venom injected, the zone of inoculation, and the species. In the Chihuahuan desert, the most abundant species is L. apachea. Its venom and biological effects are understudied, including neurological effects. Thus, our aim is to explore the effect of this regional species of medical interest in the United States-Mexico border community, using rat blood and central nervous system (CNS), particularly, two brain structures involved in brain homeostasis, Area postrema (AP) and Choroid plexus (PC). L. apachea specimens were collected and venom was obtained. Different venom concentrations (0, 0.178 and 0.87 μg/g) were inoculated into Sprague-Dawley rats (intraperitoneal injection). Subsequently, blood was extracted and stained with Wright staining; coronal sections of AP were obtained and stained with Hematoxylin-Eosin (HE) staining and laminin γ immunolabelling, the same was done with CP sections. Blood, AP and CP were observed under the microscope and abnormalities in erythrocytes and fluctuation in leukocyte types were described and quantified in blood. Capillaries were also quantified in AP and damage was described in CP. L. apachea venom produced a segmented neutrophil increment (neutrophilia), lymphocyte diminishment (leukopenia) and erythrocytes presented membrane abnormalities (acanthocytosis). Extravasated erythrocytes were observed in HE stained sections from both, AP and CP, which suggest that near to this section a hemorrhage is present; through immunohistofluorescence, a diminishment of laminin γ was observed in AP endothelial cells and in CP ependymal cells when these structures were exposed to L. apachea venom. In conclusion, L. apachea venom produced leukopenia, netrophilia and acanthocytosis in rat peripheral blood, and also generated hemorrhages on AP and CP through degradation of laminin γ.

El desarrollo económico y social de las poblaciones conlleva a la necesidad de mejorar los sistemas de abastecimiento de alimentos. Por un lado, el desarrollo agrícola y pecuario demanda un incremento en la producción con el objetivo de obtener mayores ganancias, y satisfacer, al mismo tiempo los requerimientos nutricionales necesarios de la población. Esto ha requerido del uso de agroquímicos con el propósito de aumentar la producción. El mal manejo de las prácticas de la aplicación de agroquímicos puede ocasionar serios problemas a la salud de la población ocupacionalmente expuesta. DOI: https://doi.org/10.54167/tecnociencia.v1i3.55

The plasma membrane Ca²⁺-ATPase (PMCA) removes Ca²⁺ from the cytosol into the extracellular space. Its catalytic activity can be stimulated by calmodulin (CaM) or by limited proteolysis. We evaluated the effect of chlorpromazine (CPZ) and dimethyl sulfoxide (DMSO) over the hydrolytic activity of PMCA. Activity was monitored in three different forms: native, CaM-activated and proteolyzed by trypsin. CPZ appears to inhibit PMCA without directly interfering with the C-terminal site, since it is affected by CaM and proteolysis. Although the treatment of PMCA with trypsin and CaM produces an activation, it also produces an enzymatic form that is more sensitive to inhibition by CPZ. The same case was observed in the DMSO inhibition experiments. In the absence of CPZ, DMSO produces a progressive loss of activity, but in the presence of CPZ the profile of activity against DMSO changes and produces a recovery of activity, indicating a possible partition of CPZ by the solvent. Increasing Ca²⁺ concentrations indicated that CPZ interacts with PMCA rather than with CaM. This observation is supported by docking analysis that suggests that the CPZ-PMCA interaction is non-competitive. We propose that CPZ interacts with the state of lower affinity for Ca^{2 +}.

Globally, snake envenomation is a well-known cause of death and morbidity. In many cases of snakebite, myonecrosis, dermonecrosis, hemorrhage and neurotoxicity are present. Some of these symptoms may be provoked by the envenomation itself, but others are secondary effects of the produced oxidative stress that enhances the damage produced by the venom toxins. The only oxidative stress effect known in blood is the change in oxidation number of Fe (from ferrous to ferric) in hemoglobin, generating methemoglobin but not in other macromolecules. Currently, the effects of the overproduction of methemoglobin derived from snake venom are not extensively recorded. Therefore, the present study aims to describe the oxidative stress induced by Crotalus molossus nigrescens venom using erythrocytes. Human erythrocytes were washed and incubated with different Crotalus molossus nigrescens venom concentrations (0–640 μg/mL). After 24 h, the hemolytic activity was measured followed by attenuated total reflectance-Fourier transform infrared spectroscopy, non-denaturing PAGE, conjugated diene and thiobarbituric acid reactive substances determination. Low concentrations of venom (40 μg/mL). This substance is not degraded by proteases present in the venom. By infrared spectroscopy, starting in 80 μg/mL, we observed changes in bands that are associated with protein damage (1660 and 1540 cm−1) and lipid peroxidation (2960, 2920 and 1740 cm−1). Lipid peroxidation was confirmed by conjugated diene and thiobarbituric acid reactive substance determination, in which differences were observed between the control and erythrocytes treated with venom. Crotalus molossus nigrescens venom provokes hemolysis and oxidative stress, which induces methemoglobin formation, loss of protein structure and lipid peroxidation.

The Mexican black-tailed rattlesnake Crotalus molossus nigrescens is distributed in the Mexican plateau. Its venom is known to cause hemolysis and presents fibrinogen coagulase, collagenase and fibrinolytic activities. These activities may be associated with hemostatic alterations, such as platelet aggregation, hemolysis and fibrinolysis, often described in ophidic accidents. However, the mechanisms of action of the C. m. nigrescens venom remain unclear. In this study we investigated the in vitro hemotoxic, neurotoxic, and vasculotoxic effects of the venom. We found that this venom produces two types of hemolytic responses, Oxyhemoglobin release and Methemoglobin formation. As a result of the cytotoxicity to endothelial cells produces morphological biphasic toxicity. The first step in this process is characterized by morphological changes, as well as the loss of cellular adhesion and reduction in thickness. The second phase is characterized by massive cellular aggregation and death. It also induced laminin, type IV collagen, perlecan and nidogen degradation. However, the venom did not modulate the muscular fetal and neuronal nicotinic acetylcholine receptors activity. Thus, we concluded that the C. m. nigrescens venom produced hemolysis and hemorrhages via degradation of the basement membrane components and endothelial cell cytotoxicity, but not by neurotoxicity at the receptor level in nicotinic acetylcholine receptors.

C6 glioma cells have been proposed as a cell model system to study regulation of endogenously-expressed glycine transporter 1 (GlyT1). However, there are no reports demonstrating expression of glycine transporter 2 (GlyT2) in this cell line. To explore this possibility, immunoprecipitation, western blotting, confocal microscopy, as well as glycine uptake assays using selective inhibitors to GlyT1 or GlyT2 were employed. Glycine uptake assays using a selective inhibitor against GlyT2 provided evidence that this transporter could also be expressed in this cell line. Immunoprecipitation, immunoblotting, and confocal microscopic analysis also showed presence of both GlyT1 and GlyT2 in this cell line. Taken together, results provide evidence that in addition to GlyT1, GlyT2 is also endogenously expressed in C6 glioma cells. Results presented here will help researchers to carefully evaluate use of this cell line in studies involving GlyT1.