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Collapsin Response Mediator Protein 2 (CRMP2) is an intracellular protein involved in axon and dendrite growth and specification. In this study, CRMP2 was identified in a conditioned media derived from degenerated sciatic nerves (CM). On cultured rat hippocampal neurons, acute extracellular application of CM or partially purified recombinant CRMP2 produced an increase in cytoplasmic calcium. The increase in cytoplasmic calcium was mostly mediated through NMDA receptors, with a minor contribution of N-type VDCC, and it was maintained as long as CM was present. By using live-labeling of CRMP2, Ca2+ channel binding domain 3 (CBD3) peptide derived from CRMP2, and recombinant CRMP2, we demonstrated that that this effect was mediated by an action on the extracellular side of the NMDA receptor. This is the first report of an extracellular action of CRMP2. Prolonged exposure to extracellular CRMP2, may contribute to neuronal calcium dysregulation and neuronal damage. Fil: Castillo Soto, Francisca Cecilia. Instituto de Estudios Avanzados ; Venezuela Fil: Martinez Rozas, Juan Carlos. Instituto de Estudios Avanzados ; Venezuela Fil: Longart, Marines. Instituto de Estudios Avanzados ; Venezuela Fil: García, Lisbeth. Instituto de Estudios Avanzados ; Venezuela Fil: Hernández, Marianela. Instituto de Estudios Avanzados ; Venezuela Fil: Carballo, Jeismar. Instituto de Estudios Avanzados ; Venezuela Fil: Rojas, Héctor Gustavo. Universidad Central de Venezuela. Facultad de Medicina; Venezuela Fil: Matteo, Lorena Romina. Instituto de Estudios Avanzados ; Venezuela Fil: Casique, Liliana. Universidad Simon Bolivar.; Venezuela Fil: Escalona Hernández, José Leonardo. Universidad Simon Bolivar.; Venezuela Fil: Rodríguez, Yuryanni. Instituto de Estudios Avanzados ; Venezuela Fil: Rodríguez Durán, Jessica Jenireth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Estudios Avanzados ; Venezuela Fil: Hernández, Deyanell. Instituto de Estudios Avanzados ; Venezuela Fil: Balbi, Domingo. Instituto de Estudios Avanzados ; Venezuela Fil: Villegas, Raimundo. Instituto de Estudios Avanzados ; Venezuela

In this work the kinematics of a three-legged parallel manipulator with asymmetrical limbs and decoupled motions over its moving platform is investigated by means of the theory of screws. The solution of the forward displacement analysis (FDA) is presented in echelon-form solution using a novel procedure based on simple geometric constraints, thus all the feasible locations of the moving platform, with respect to the fixed platform, are computed given a set of generalized coordinates. After, the velocity and acceleration analyses are carried out using the theory of screws. Finally, a numerical example is included and the results obtained via the screw theory are verified with the aid of special commercially available software like MSC.Adams©.

Rotavirus infection is characterized by a number of Ca 2+ dependent virus–cell interactions. The structure of rotavirus triple-layered particles (TLP) is dependent on Ca 2+ concentration. Acquisition of the capsid outer layer requires a high Ca 2+ concentration inside the ER. Infection modifies Ca 2+ homeostasis of the cell, increasing ER Ca 2+ content, which may be advantageous to virus replication. We studied the role of sequestered Ca 2+ on the stabilization of already mature viral particles within the ER. Thapsigargin (TG), a SERCA pump inhibitor, added for 30 min at the end of infection depleted ER Ca 2+ and reduced the titer of already mature TLP accumulated in the cell. Another inhibitor, cyclopiazonic acid, and two Ca 2+ ionophores (A23187 and ionomycin) in the presence of EGTA had similar effects. TG eliminated the peak of radiolabeled TLP, increasing that of DLP in CsCl gradients. Electron microscopy revealed accumulation of clustered particles in the ER, which had lost their integrity. The [Ca 2+ ] in the ER of infected cells is important for virus maturation and for maintaining the integrity of mature TLP. Viral particles in this compartment may be potentially infectious, already containing VP7 and VP4.

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.

Grafting of polyethylenes is an important method used in the modification of polyolefins with functional groups. Nevertheless, few systematic studies have been conducted regarding this type of reactions and the interdependent variables are not yet accurately established. In this investigation, the grafting of two commercial polyethylenes, low-density polyethylene and linear low-density polyethylene with diethyl maleate was carried out in a corotating twin-screw extruder at different extrusion conditions. In order to establish the interdependence of the extrusion conditions employed, a combined experimental and simplified one-dimensional, non-isothermal model was implemented in the study of flow in the elements of the screw-extruder. The effects of the grafting degree and the processing conditions employed in the preparation of the functionalized materials on their shear and elongational viscosities at high shear rates, and on their thermal and tensile properties were studied.

A new Ca2+ intracellular store, the acidocalcisome, has been reported in trypanosomatids. It has been characterized physiologically as a Ca2+ store sensitive to nigericin. The Ca2+/H+-ATPase is the system responsible for Ca2+ accumulation, which depends on a pH gradient formed by ATP- and PPi-dependent proton pumps. In this work we present physiological and morphological evidences for the presence of acidocalcisomes in Trypanosoma evansi. The parasites were purified and loaded with the fluorescent dye Fura 2-AM in order to detect the intracellular changes of Ca2+ levels in individual cells. The simultaneous incubation of T. evansi cells with ionomycin and nigericin led to large release of Ca2+ (ca. 200 nM) from intracellular stores, which was not observed with either agent alone. On the other hand, no enhancement of the nigericin-induced Ca2+ release was observed in the presence of oligomycin. Additionally, the pretreatment with bafilomycin decreases the nigericin-induced Ca2+ release. These results confirm the presence of an intracellular non-mitochondrial acidic Ca2+ storage compartment. These results suggest that H+-ATPase is involved in the process of Ca2+ accumulation into the acidocalcisomes. Furthermore, the cells loaded with acridine orange exhibited abundant fluorescent vacuoles, which were sensitive to nigericin or bafilomycin A1. Electronic transmission microscopy observations demonstrated the presence of electron dense particles in the parasites. High levels of inorganic pyrophosphate and triphosphate were detected in perchloric acid extracts of T. evansi by high resolution 31P NMR. Taken together, these results present the first evidence for the presence of acidocalcisomes in T. evansi.

Ca2+-selective electrodes have been used to measure free intracellular Ca2+ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 microns in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca2+ down to about 3 microM in solutions containing 0.3 M K+ and 0.025 M Na+. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca2+ over Mg2+, H+, K+ and Na+. To calibrate them properly, a set of standard solutions were prepared using different Ca2+ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca2+ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca2+, the mean resting intracellular ionized calcium concentration was 0.106 microM (n = 15). The Ca2+-electrodes were used to investigate effects of different experimental procedures on the [Ca2+]i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca2+]i levels by a process independent of external Na+; (ii) poisoned axons can extrude calcium ions at high levels of [Ca2+]i by an external Na+-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.