Ca2+]i regulates trafficking of Cav1.3 (alpha1D Ca2+ channel) in insulin-secreting cells

Chronic exposure of pancreatic -cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of -cell basal intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We have investigated the relationship between the basal intracellular free Ca²⁺ and the L-type (Ca_v1.3) Ca²⁺ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca²⁺ influx, which was attenuated when the basal [Ca²⁺]_i was elevated. Using voltage-clamp techniques, we found that changing [Ca²⁺]_i could regulate the amplitude of the Ca²⁺ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Ca_v1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca²⁺]_i conditions by deconvolution microscopy. The [Ca²⁺]_i-dependent translocation of Ca_v1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca²⁺]_i. These results suggest that Ca_v1.3 channel trafficking is involved in glucose desensitization of pancreatic -cells. internalization; intracellular free calcium; glucose desensitization     

Seasonal variation in the diet of wild rabbits<Emphasis Type="Italic">Oryctolagus cuniculus</Emphasis> on a semiarid Atlantic island (Alegranza,Canarian Archipelago)

The diet of the wild rabbitOryctolagus cuniculus (Linnaeus, 1758) on Alegranza Island (Canary Islands) was studied by microhistological examination of faecal samples. We compared the presence of each food-plant species in the faeces across a year in comparison with plant availability. Two areas were studied, Llano de la Vega and La Caldera. At the first site,Suaeda vera andChenoleoides tomentosa were the most common plants, while at the latter it wasEuphorbia regis-jubae. In general, species belonging to the Chenopodiaceae were most commonly eaten by rabbits. Plants such asChenoleoides tomentosa andSalsola vermiculata were most important in the diet in every season. Levin’s niche-breadth was broader across the seasons in the study site of La Caldera than at Llano de la Vega. Furthermore, this parameter yielded the highest values (broadest niche) during the spring at both sites. Most important plants eaten by rabbits showed green vegetative parts through all seasons. Rabbits selected some plants independently of their availability, even in this reduced insular environment. The feeding behaviour of rabbits in Alegranza, selecting certain species, must have had important consequences on the composition and distribution of the vegetation over the years. This negative effect on island vegetation has been shown to occur on many oceanic islands of the world where rabbits have been introduced.     

A very sensitive enzyme-linked immunoabsorbent assay to Staphylococcal protein A in the presence of immunoglobulins

The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)--prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)(2) (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.     

Bioprocess applications of a Sindbis virus-based temperature-inducible expression system

The production and study of toxic proteins requires inducible expression systems with low basal level expression and high inducibility. Here, we describe bioprocess applications of the pCytTS temperature-regulatable Sindbis virus replicon-based expression system. We used green fluorescent protein as a marker protein to optimize the selection of stable transfected clones with increased expression levels. Using the optimized protocol, clones were constructed that produced the growth-inhibiting, anti-viral protein interferon beta (beta-IFN). Selected clones were analyzed for temperature-dependent beta-IFN production in adherent and suspension cultures in serum free medium. Specific expression levels were around 1.0 x 10(5) IU/10(6) cells/day (0.5 microg/10(6) cells/day) in suspension cultures and over 1.5 x 10(6) IU/mL/day (7.5 microg/mL/day) in hollow fiber reactors using adherent cells. Hexahistidine-tagged beta-IFN purified from T-flask cultures was highly glycosylated and showed high specific activity. beta-IFN mRNA amplified by the viral replicase for 10 days did not show an accumulation of mutations. These data suggest the applicability of the pCytTS-inducible expression system for the production of high-quality glycoproteins in different reactors.     

Different patterns of retinal cone topography in two genera of rodents,Mus and Apodemus

Recently, we have reported the peculiar topographic separation of shortwave- and middlewave-sensitive (S and M) cones in the retina of the common house mouse (Mus musculus) and in a number of inbred laboratory mouse strains derived from the same species. In an attempt to follow the phylogeny of the complementary cone fields, we have investigated the retina of other mouse-like rodents. Two monoclonal anti-visual pigment antibodies, OS-2 and COS-1, specific to the S and M cones, respectively, have been used to identify the two cone types. Immunocytochemistry on retinal sections and on whole-mount preparations have shown that, as in the house mouse, the two cone types in the mound builder mouse (Mus spicileugus) occupy opposite halves of the retina. In contrast, in the wood mouse (Apodemus sylvaticus), both cone types are scattered uniformly across the whole retinal surface. Another distinguishing feature between the two genera is the frequency of the S cones. Whereas their density in the Mus species is above 7 000/mm² in the S-field, the maximum density of the S cones in A. sylvaticus is one order of magnitude smaller. In another species of this genus (the herb field mouse, A. microps), the S cones are completely missing.