Sphingomyelinase D Activity in Model Membranes: Structural Effects of in situ Generation of Ceramide-1-Phosphate

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes

Effectiveness of two common antivenoms for North, Central, and South American Micrurus envenomations

Micrurus snakes (coral snakes) may produce severe envenomation that can lead to death by peripheral respiratory paralysis. Only few laboratories produce specific antivenoms, and despite the cross-reactivity found in some Micrurus species venoms, the treatment is not always effective. To test two therapeutic antivenoms against the venom of four species of Micrurus from Southern America, North of South America, Central America, and North America, the determination of the lethal potency of the venoms, the study of some biochemical and immunochemical characteristics, and the determination of the neutralizing activity of both antivenoms were studied. North American and South American antivenoms neutralized well venoms from Micrurus species of the corresponding hemisphere but displayed lower effectiveness against venoms of species from different hemispheres. It was concluded that the neutralization of Micrurus venoms by regional antivenoms could be useful to treat the envenomation by some Micrurus snakes but is necessary to evaluate carefully the antivenoms to be used with the venoms from the snakes of the region. Also, considering the difficulties for coral snake antivenom production, the development of a polyvalent antivenom is useful to treat the envenomation by coral snakes from different regions is necessary.Fil: de Roodt, Adolfo R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Área de Investigación y Desarrollo. Serpentario; Argentina.Fil: Paniagua-Solís, Jorge F. Laboratorios Silanes S.A. de C.V. Dirección de Investigación y Desarrollo; México.Fil: Dolab, Jorge A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Departamento de Vacunas y Sueros; Argentina.Fil: Estevez-Ramirez, Judith. Instituto Bioclón. Laboratorio Investigación y Desarrollo; México.Fil: Ramos-Cerrillo, Blanca. Universidad Autónoma de México (UNAM). Departamento de Medicina Molecular y Bioprocesos; México.Fil: Litwin, Silvana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Área de Investigación y Desarrollo. Serpentario; Argentina.Fil: Dokmetjian, J. C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Área de Investigación y Desarrollo. Serpentario; Argentina.Fil: Alagón, Alejandro. Universidad Autónoma de México (UNAM). Departamento de Medicina Molecular y Bioprocesos; México

Effectiveness of two common antivenoms for North, Central, and South American Micrurus envenomations

Micrurus snakes (coral snakes) may produce severe envenomation that can lead to death by peripheral respiratory paralysis. Only few laboratories produce specific antivenoms, and despite the cross-reactivity found in some Micrurus species venoms, the treatment is not always effective. To test two therapeutic antivenoms against the venom of four species of Micrurus from Southern America, North of South America, Central America, and North America, the determination of the lethal potency of the venoms, the study of some biochemical and immunochemical characteristics, and the determination of the neutralizing activity of both antivenoms were studied. North American and South American antivenoms neutralized well venoms from Micrurus species of the corresponding hemisphere but displayed lower effectiveness against venoms of species from different hemispheres. It was concluded that the neutralization of Micrurus venoms by regional antivenoms could be useful to treat the envenomation by some Micrurus snakes but is necessary to evaluate carefully the antivenoms to be used with the venoms from the snakes of the region. Also, considering the difficulties for coral snake antivenom production, the development of a polyvalent antivenom is useful to treat the envenomation by coral snakes from different regions is necessary.Fil: de Roodt, Adolfo R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Área de Investigación y Desarrollo. Serpentario; Argentina.Fil: Paniagua-Solís, Jorge F. Laboratorios Silanes S.A. de C.V. Dirección de Investigación y Desarrollo; México.Fil: Dolab, Jorge A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Departamento de Vacunas y Sueros; Argentina.Fil: Estevez-Ramirez, Judith. Instituto Bioclón. Laboratorio Investigación y Desarrollo; México.Fil: Ramos-Cerrillo, Blanca. Universidad Autónoma de México (UNAM). Departamento de Medicina Molecular y Bioprocesos; México.Fil: Litwin, Silvana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Área de Investigación y Desarrollo. Serpentario; Argentina.Fil: Dokmetjian, J. C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos. Área de Investigación y Desarrollo. Serpentario; Argentina.Fil: Alagón, Alejandro. Universidad Autónoma de México (UNAM). Departamento de Medicina Molecular y Bioprocesos; México

Effect of product on relative enzyme specific activity and on vesicle size.

<p>A) Specific activity of SMD on C₁₂SM LUVs with increasing molar fractions of C₁₂Cer-1-P. With the exception of pure C₁₂SM and the 50 mol% mixture, all other differences are statistically significant (P<0.003). B) Mean diameter in nm versus time obtained by DLS of C₁₂SM∶C₁₂Cer-1-P LUVs: The black open circles represent pure C₁₂SM untreated vesicles, the green triangles represent untreated C₁₂SM∶C₁₂Cer-1-P vesicles (23 mol%), the closed red squares are C₁₂SM∶C₁₂Cer-1-P vesicles (50 mol%) treated with 1 µg/ml SMD and the open red squares are the same but without enzyme.</p

Representative fluorescence images of the effect of SMD on GUVs composed of DOPC∶eggSM∶cholesterol 2∶1∶1 mol.

<p>A) Domains in a DiIC₁₈-labeled GUV exposed to inactive Lb3 for 16 hours. B) Representative DiIC₁₈-labeled GUVs before and after treatment with SMD. C) Two LAURDAN-labeled GUVs before (left) and after (right) exposure to SMD. Before treatment two domains are apparent (average GP values of 0.1 and 0.5 indicated by the green and red arrows, respectively), after SMD action the membrane becomes uniform (average GP value of 0.3). Bars are 5 µm.</p

Protein fluorescence spectra of SMD.

<p>The main panel shows the normalized fluorescence emission spectrum of SMD (excitation at 280 nm). The black trace is SMD alone, mixed with POPC vesicles (green) and with C₁₂SM LUVs (red). Inset: fluorescence polarization values for the same samples. The differences in polarization are statistically significant (P<0.003).</p

Effect of cholesterol on relative enzyme specific activity and on vesicle size.

<p>A) Effect of cholesterol on the specific activity of SMD. With the exception of pure C₁₂SM and the 9 mol% mixture, all other differences are statistically significant (P<0.003). B) Mean diameter in nm versus time obtained by DLS of C₁₂SM∶Cholesterol LUVs: The black circles represent pure C₁₂SM vesicles with SMD, in green C₁₂SM∶cholesterol vesicles (23 mol%) with (closed squares) and without (open squares) SMD and in red C₁₂SM∶cholesterol vesicles (50 mol%) with SMD (closed triangles) and without (open triangles). Enzyme concentration was 1 µg/ml.</p

Fluorescence correlation spectroscopy of C₁₂SM LUVs under the action of SMD.

<p>A) Representative normalized autocorrelation plots for the untreated vesicles (green circles), the same preparation treated with 1 µg/ml SMD (red circles) and a control with Triton X-100 (open black circles). B) Diffusion coefficient (D₂) in time calculated from fitting the autocorrelation plot for the enzyme-treated sample. The bottom panels represent fluorescence images taken at 24 hours of C) sedimented material after enzyme treatment (1 µg/ml SMD) and D) untreated C₁₂SM LUVs. The images are 30 µm×30 µm.</p