Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.     

Stevens-Johnson Syndrome Associated with Drugs and Vaccines in Children: A Case-Control Study

Objective

Stevens-Johnson Syndrome (SJS) is one of the most severe muco-cutaneous diseases and its occurrence is often attributed to drug use. The aim of the present study is to quantify the risk of SJS in association with drug and vaccine use in children.

Methods

A multicenter surveillance of children hospitalized through the emergency departments for acute conditions of interest is currently ongoing in Italy. Cases with a diagnosis of SJS were retrieved from all admissions. Parents were interviewed on child’s use of drugs and vaccines preceding the onset of symptoms that led to the hospitalization. We compared the use of drugs and vaccines in cases with the corresponding use in a control group of children hospitalized for acute neurological conditions.

Results

Twenty-nine children with a diagnosis of SJS and 1,362 with neurological disorders were hospitalized between 1^st November 1999 and 31^st October 2012. Cases were more frequently exposed to drugs (79% vs 58% in the control group; adjusted OR 2.4; 95% CI 1.0–6.1). Anticonvulsants presented the highest adjusted OR: 26.8 (95% CI 8.4–86.0). Significantly elevated risks were also estimated for antibiotics use (adjusted OR 3.3; 95% CI 1.5–7.2), corticosteroids (adjusted OR 4.2; 95% CI 1.8–9.9) and paracetamol (adjusted OR 3.2; 95% CI 1.5–6.9). No increased risk was estimated for vaccines (adjusted OR: 0.9; 95% CI 0.3–2.8).

Discussion

Our study provides additional evidence on the etiologic role of drugs and vaccines in the occurrence of SJS in children.     

A histochemical study of the binding of 125I-HCG to the rat ovary throughout the estrous cycle

Summary Changes in the distribution of the in vitro uptake of ¹²⁵I-HCG by the ovaries of adult rats were examined histochemically throughout the estrous cycle.Only in follicles wider than 500 m, occurring mainly at diestrus and proestrus, could granulosa cells bind the labelled hormone. The labelling increased with follicular size and decreased in intensity from the peripheral granulosa cells inwards. No uptake occurred in the oocytes, in the cells of the cumulus oophorus nor in the granulosa cells of the atretic follicles.The binding capacity of the newly-formed corpora lutea of estrus was less than that of preovulatory follicles. The uptake of ¹²⁵I-HCG by corpora lutea during the first cycle reached its maximum at diestrus but fell sharply by proestrus. The uptake was patchy in the corpora lutea of the second cycle and not significant in the older ones.The uptake of ¹²⁵I-HCG by thecae increased with follicular size and was greater in the thecae of atretic follicles than in the thecae of growing follicles of like size. There was a greater uptake in the last formed interstitial tissue than there was in older tissue.At proestrus, the uptake of ¹²⁵I-HCG was unaffected by the LH surge at 18.00h but had decreased slightly at 24.00 h.The implications of these data in relation to the regulation of receptor sites, is discussed.     

SNAP-25 and synaptotagmin 1 function in Ca2+-dependent reversible docking of granules to the plasma membrane

In neuroendocrine cells, Ca²⁺ triggers fusion of granules with the plasma membrane and functions at earlier steps by increasing the size of the readily releasable pool of vesicles. The effect of Ca²⁺ at early steps of secretion may be due to the recruitment at the plasma membrane of granules localized in the cytoplasm. To study the mechanism of granule docking, a new in vitro assay is designed using membrane fractions from mouse pituitary AtT-20 cells. By using this assay, it is found that granule docking to the plasma membrane is controlled by Ca²⁺ concentrations in the micromolar range, is reversible and requires intact SNAP-25, but not VAMP-2. In the docking assay, addition of Ca²⁺ induces the formation of a SNAP-25-Synaptotagmin 1 complex. The cytosolic domain C2AB of Synaptotagmin 1 and anti-Synaptotagmin 1 antibodies block granule docking. These results show that Ca²⁺ modulates dynamic docking of granules to the plasma membrane and that this process is due to a Ca²⁺-dependent interaction between SNAP-25 and Synaptotagmin 1 .     

Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.     

Estimating the long-term repeatability of food-hoarding behaviours in an avian predator

Food-hoarding behaviour is widespread in the animal kingdom and enables predictable access to food resources in unpredictable environments. Within species, consistent variation among individuals in food-hoarding behaviours may indicate the existence of individual strategies, as it likely captures intrinsic differences in how individuals cope with risks (e.g. starvation, pilferage). Using 17 years of data, we estimated the long-term repeatability of 10 food-hoarding behaviours in a population of Eurasian pygmy owls (Glaucidium passerinum), a small avian predator subject to high temporal fluctuations in its main prey abundance. We found low repeatability in the proportion of shrews and the average prey mass stored for both sexes, while females were moderately repeatable in the mass and the number of prey items stored. These two pairs of behaviours were tightly correlated among individuals and might represent two different sets of individual strategies to buffer against starvation risks.     

Isolation, heterologous expression and characterization of an endo-polygalacturonase produced by the phytopathogen Burkholderia cepacia

Endo-polygalacturonases (endoPGs) belong to the glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond present in the smooth regions of pectins. Pectic substances are among the principal macromolecular components of the primary plant cell walls and are subjected to enzymatic degradation not only in the course of important physiological processes such as plant senescence and ripening, but also during infection events by plant pathogens. Here we report, for the first time, the isolation and the purification of an endoPG (PehA) from the supernatant of the plant pathogen Burkholderia cepacia strain ATCC 25416. In order to obtain adequate amounts of protein required for structural and functional studies, the gene coding for pehA was PCR-amplified and cloned in Escherichia coli cells. The recombinant protein was purified to homogeneity and characterized. PehA exhibited a pI value of 8.0 and an optimal activity at pH 3.5. Far-UV circular dichroism (CD) measurements show that PehA assumes a beta-helix fold super-secondary structural motif.