Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.     

Ecology, inhibitory activity, and morphogenesis of a marine antagonistic bacterium belonging to the Roseobacter clade

Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30 degrees C but died rapidly at 37 degrees C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37 degrees C in less than 2 days and at 25 degrees C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.     

The Main Regulatory Region in the Murine PSP Gene is a Parotid Gland Enhancer

The murine PSP gene is expressed at a high-level in the parotid glands. To extend the knowledge of parotid gland expression and develop tools for expression of heterologous proteins in this tissue, the regulation of the PSP gene was studied using transgenic mice. High-level parotid gland expression of the PSP gene was indicated to depend on a novel regulatory region situated between –8.0 and –6.5 kb. Together with previous results this indicates that the main regulatory elements in the PSP gene are situated between –8.0 to –3.1 kb. This region was shown to activate a heterologous SV40 early promoter in the parotid glands of transgenic mice, suggesting that the PSP gene is controlled by enhancer sequences. A novel Psp derived 9.7 kb parotid gland expression cassette, Lama IV, carrying all known regulatory regions in the PSP gene was expressed at high-levels in the parotid glands and should prove highly useful for expression of heterologous proteins in the saliva of transgenic mice.     

Ecto-ATP diphosphohydrolase/CD39 is overexpressed in differentiated human melanomas

Ecto-ATPase activities of melanocytes and human melanoma cell lines differing in the stage of progression were compared. A dramatic increase in ecto-ATPase activity above the level of normal melanocytes was demonstrated in the differentiated melanomas and was followed by a gradual decrease with tumor progression. The characteristics of this enzymatic activity were consistent with CD39/ecto-ATP diphosphohydrolase (ATPDase) which was found to be the major ecto-ATP-hydrolysing enzyme in melanomas. Indeed, the expression of CD39 and the level of CD39 mRNA followed a similar pattern. Since CD39 is known to regulate homotypic adhesion and, supposedly, affects the disaggregation step, we suggest that overexpression of CD39 may enable tumor cells to reduce contacts with T-lymphocytes and escape from immunological recognition.     

Induction of HLA expression in Daudi cells after cell fusion

The Daudi cell line, established from a Burkitt lymphoma, has recently been found to be HLA- andΒ ₂-microglobulin-negative, although it expresses B lymphocyte alloantigens. This report is concerned with the reexpression of HLA-A10, B38, and B17 on the Daudi cell, after cell fusion with another human cell line (Raji) or with mouse fibroblasts. In the latter fusion, the same HLA specificities are re-expressed, but not humanΒ ₂-microglobulin while mouseΒ ₂-microglobulin andH-2 could be detected. No such reexpression was observed when Daudi was fused with the F9 mouse teratocarcinoma, which lacks mouseΒ ₂-m andH-2. No HLA activity (alloantigenic and xenogenic activity) was detected in the membrane or cytoplasm of Daudi, using salt extraction and sonication. Therefore we postulate thatΒ ₂-microglobulin could be necessary for the expression and possible synthesis of the HLA antigen.     

Comparative immunocytochemical localization of prolactin and somatotropin in the pituitaries of Lepidosiren paradoxa,Rana temporaria and Ambystoma mexicanum

Summary The cellular binding sites of anti-oPRL IgG and anti-bSTH IgG were demonstrated in the pituitary glands of Lepidosiren paradoxa, Rana temporaria and Ambystoma mexicanum by means of the unlabeled antibody enzyme method by light and electron microscopy (the latter only in Lepidosiren). With the light microscope PRL or PRL-like substances and STH or STH-like substances were revealed in two different cell types in the distal lobe corresponding to the acidophils. However, as a result of the insufficient differentiation of the acidophils in Lepidosiren after staining with Brookes' procedure it was not possible to distinguish the two types of acidophils in this animal. Treatment with low dilutions of both anti-oPRL and anti-bSTH IgG revealed simultaneous immunocytochemical staining in both types of acidophils in Lepidosiren and in Rana. These results, indicating that there is antigenic cross-reaction between anti-oPRL and anti-bSTH IgG and both PRL and STH in these animals, are discussed.The electron microscopic investigations of Lepidosiren revealed that the specific anti-oPRL IgG reactive cells contain granules ranging in size from 200 to 300 nm, while the specific anti-bSTH IgG reactive cells contain smaller immunoreactive granules ranging from 80 to 160nm.     

The −14010*C variant associated with lactase persistence is located between an Oct-1 and HNF1α binding site and increases lactase promoter activity

In most people worldwide intestinal lactase expression declines in childhood. In many others, particularly in Europeans, lactase expression persists into adult life. The lactase persistence phenotype is in Europe associated with the −13910*T single nucleotide variant located 13,910 bp upstream the lactase gene in an enhancer region that affects lactase promoter activity. This variant falls in an Oct-1 binding site and shows greater Oct-1 binding than the ancestral variant and increases enhancer activity. Several other variants have been identified very close to the −13910 position, which are associated with lactase persistence in the Middle East and Africa. One of them, the −14010*C, is associated with lactase persistence in Africa. Here we show by deletion analysis that the −14010 position is located in a 144 bp region that reduces the enhancer activity. In transfections the −14010*C allele shows a stronger enhancer effect than the ancestral −14010*G allele. Binding sites for Oct-1 and HNF1α surrounding the −14010 position were identified by gel shift assays, which indicated that −14010*C has greater binding affinity to Oct-1 than −14010*G.     

Gender difference in antidiuretic response to desmopressin

Increased age and female gender are well-known risk factors for the development of desmopressin-induced hyponatremia. However, little focus has been on exploring gender differences in the antidiuretic response to desmopressin. Based on an exploratory analysis from three clinical trials, we report a significant gender difference in the effects of desmopressin on nocturnal urine volume that could not be explained by pharmacokinetic differences. Mean desmopressin concentration profiles were tested for covariates, and age and gender were not statistically significant and only weight was significant for log(C(max)) (P = 0.0183) and borderline significant for log(AUC) (P = 0.0571). The decrease in nocturnal urine volume in nocturia patients treated with desmopressin over 28 days was significantly larger for women at the lower desmopressin melt doses of 10 and 25 μg than for men. The ED(50) for men was modeled to be 43.2 μg and 16.1 μg for women, with the ED(50) men/women estimated to be 2.7 (1.3-8.1 95% CI), corresponding to significantly higher sensitivity to desmopressin in women. An increasing incidence of hyponatremia with increasing dose was found, and at the highest dose level of 100 μg decreases in serum sodium were approximately twofold greater in women over 50 yr of age than in men. A new dose recommendation stratified by gender is suggested in the treatment of nocturia: for men, 50- to 100-μg melt is an efficacious and safe dose, while for women a dose of 25 μg melt is recommended as efficacious with no observed incidences of hyponatremia. Areas for further research are proposed to uncover pathophysiological mechanism(s) behind these gender differences.     

Proteomic and immunoproteomic analysis of Aggregatibacter actinomycetemcomitans JP2 clone strain HK1651

The proteome of Aggregatibacter actinomycetemcomitans HK1651 (JP2 clone) and immunoreactive antigens were studied by two-dimensional (2D) gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and 2D immunoblotting. The highly leukotoxic JP2 clone of A. actinomycetemcomitans is strongly associated with aggressive periodontitis (AgP) in adolescents of North-West African descent and the pathogenicity of this bacterium is of major interest. Hence, we developed a comprehensive 2D proteome reference map of A. actinomycetemcomitans proteins with 167 identified spots representing 114 different proteins of which 15 were outer membrane proteins. To unravel immunoreactive antigens, we applied 2D-gel and subsequent immunoblotting analyses using sera from five individuals with A. actinomycetemcomitans infections and one healthy control. The analysis revealed 32 immunoreactive proteins. Antibodies to two outer membrane proteins, YaeT (85 kDa) and Omp39 (39 kDa), not previously described as immunoreactive, were found only in subjects with current or previous A. actinomycetemcomitans JP2 infection. Further proteome-based studies of A. actinomycetemcomitans combined with analyses of the humoral immune response and targeted against outer membrane proteins may provide important insight into the host relationship of this important pathogen.