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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 methanol...

The venom of

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.

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.

Resumen es: Los envenenamientos por mordeduras de serpientes de cascabel son de importancia medica. Los diversos factores que la medicina atiende son el clima, la ed...

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.

"Las propiedades terapéuticas del chile se atribuyen a los fitoquímicos que contiene. Los principales compuestos cuantificados en el extracto metanólico de chile jalapeño verde fueron: capsaicina (9.15 mg/g), quercetina (4.65 mg/g), carotenoides (3.68 mg equivalentes de β-caroteno/g) y ácido cafeico (0.57 mg/g). Se evaluó el efecto citotóxico de dicho extracto y de sus fitoquímicos representativos sobre adipocitos de la línea celular 3T3-L1. El mayor efecto citotóxico fue con capsaicina (IC 50 335 μg/mL) y β-caroteno (IC 50 457 μg/mL). Capsaicina combinada con el extracto u otros fitoquímicos presentó el mismo efecto citotóxico que de forma individual, en cambio, quercetina y ácido cafeico combinados con el extracto u otros fitoquímicos, presentaron un efecto citotóxico menor que de forma individual. Estos resultados proponen una alternativa terapéutica natural al tratamiento y prevención de la obesidad."

Background 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. Methods 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. Results 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. Conclusions Crotalus molossus nigrescens venom provokes hemolysis and oxidative stress, which induces methemoglobin formation, loss of protein structure and lipid peroxidation.

Background 1,2-Dichlorobenzene (1,2-DCB) is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca2+-ATPases, have been poorly studied. Therefore, we aimed to study the effect of 1,2-DCB on the Ca2+-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca2+ concentration. Results Concentrations of 0.05–0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca2+-ionophore. At higher concentrations (0.25–0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca2+ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca2+ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca2+ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-32P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C. Conclusions The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism – E2P · Ca2 state to E2 and Pi, which explains the uncoupling of the ATP hydrolysis from the Ca2+ transport.

The Northern black-tailed rattlesnake (Crotalus molossus molossus) venom is mainly hemotoxic, hemorrhagic, and neurotoxic. Its effects in the central nervous system are unknown and only poorly described for all Viperidae species in general. This is why we are interested in describe the damage induced by C. m. molossus venom in rat brain, particularly in the area postrema capillaries. Four C. m. molossus venom doses were tested (0.02, 0.05, 0.10 and 0.20mg/kg) injected intramuscularly at the lower limb, incubated by 24 hours and the brains were harvested. Area postrema coronal sections were stained with Haematoxylin and Eosin, and examined to observe the venom effect in quantity of capillaries and porphology. Starting from the 0.10mg/kg treatment we observed lysed extravasated erythrocytes and also capillary breakdown, as a consequence of hemorrhages appearance. The number of capillaries decreased significantly in response to the venom dose increment. Hemorrhages could be caused by the metalloproteinase activity on the basal membrane and the apoptosis generated by L-amino acid oxidases. Hemolysis could be caused by phospholipase A2 hemotoxic effect. We conclude that C. m. molossus crude venom produces hemolysis, capillary breakdown, hemorrhages, and the reduction in number of capillaries in the area postrema.

Microinjection through the tough vitelline coats of oocytes from three molluscan species (Haliotis rufescens, Norrisia norrisi, and Astrea undosa) was facilitated by the local appli- cation of sperm vitelline coat lysin with a micropipet. Extracts containing lysin were the 12,OOOg supernates of frozen-thawed sperm. In H. rufescens and N. norrisi, lysin extracts dissolved a small hole through the vitelline coat, whereas in A. undosa, the lysin created a pathway for mi- croinjection by softening the vitelline coat without dissolving it. Similar effects were observed when oocytes were suspended in lysin extracts: Vitelline coats dissolved completely in H. rufescens (previously reported by Lewis et al. (19821, Dev. Biol. 92:227-239) and N. norrisi, but only soft- ened and swelled in A. undosa (although they could then be removed by mechanical agitation). The lysin extracts were highly species specific and had no visible effects on heterologous vitelline coats even at concentrations higher than those required to dissolve/soften homologous coats. Evi- dence that N. norrisi lysin acts by a non-enzymatic mechanism was provided by the observation that mobilities of radioactive bands in sodium dodecyl sulfate polyacrylamide gels were unchanged following dissolution of lZ5I-labeled vitelline coats. Electron micrographs of the previously undescribed sperm from N. norrisi and A. undosa are also presented. o 1995 \ViIey-Liss, Inc. In many animal species, the exposure of lytic agents, called lysins, during the acrosome reac- tion enables sperm to penetrate through the ex- ternal coat of the egg and fuse with the egg plasma membrane (reviewed by Hoshi, '85). Among inver- tebrates, most information about lysins comes from the molluscs (Hoshi, '85). Sperm lysins that dissolve egg vitelline coats (VCs) have been ob- tained from the giant keyhole limpet Megathura crenulata (Tyler, '391, the mussells MytiZus edulis and californianus (Berg, '501, various snails (Tegula species, Haino, '71; ?2lrbo cornutus, Ogawa and Haino-Fukushima, '84), and abalone (Haliotis corrugata, cracheroderi, discus and rufescens: Tyler, '39; Lewis et al., '82; Haino-Fukushima and Usui, '86; Vacquier et al., '90). In Tegula (Haino- Fukushima, ,741, Haliotis (Lewis et al., '82), and %rho (Ogawa and Haino-Fukushima, '84), the mechanism of action of lysin is non-enzymatic: a stoichiometric combination of lysin molecules with yet-to-be-identified VC components causes the dis- solution of the VC with no evidence of covalent bond breakage. When we initiated experiments involving the microinjection of abalone oocytes, we found that it was difficult to penetrate the tough VC with- out damaging or killing the oocyte. Removal of