Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia

In Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating-competent cells with D-[6-(3)H]galactose, and then analyzed the radiolabelled proteins by anion exchange chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating-competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition assays.     

A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: comparison with the in vivo Draize rabbit test

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.     

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.     

Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A<Subscript>2B</Subscript> adenosine receptors

Adenosine A_2B receptors (A_2BR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A_2BR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A_2BR localization was examined by immunofluorescence. The role of A_2BR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A_2BR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A_2BR antagonist MRS1754 enhanced electrically induced NK₁-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A_2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A_2BR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A_2BR expression. A_2BR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.     

DNA decay rate in papyri and human remains from Egyptian archaeological sites

The writing sheets made with strips from the stem (caulis) of papyri (Cyperus papyrus) are one of the most ingenious products of ancient technology. We extracted DNA from samples of modern papyri varying in age from 0-100 years BP and from ancient specimens from Egypt, with an age-span from 1,300-3,200 years BP. The copy number of the plant chloroplast DNA in the sheets was determined using a competitive PCR system designed on the basis of a short (90 bp) tract of the chloroplast's ribulose bisphosphate carboxylase large subunit (rbcL) gene sequence. The results allowed us to establish that the DNA half-life in papyri is about 19-24 years. This means that the last DNA fragments will vanish within no more than 532-672 years from the sheets being manufactured. In a parallel investigation, we checked the archaeological specimens for the presence of residual DNA and determined the extent of racemization of aspartic (Asp) acid in both modern and ancient specimens, as a previous report (Poinar et al. [1996], Science 272:864-866) showed that racemization of aspartic acid and DNA decay are linked. The results confirmed the complete loss of authentic DNA, even in the less ancient (8th century AD) papyri. On the other hand, when the regression for Asp racemization rates in papyri was compared with that for human and animal remains from Egyptian archaeological sites, it proved, quite surprisingly, that the regressions are virtually identical. Our study provides an indirect argument against the reliability of claims about the recovery of authentic DNA from Egyptian mummies and bone remains.     

Cyclic guanosine monophosphate role in human carcinoma pathogenesis

In order to examine the cyclic nucleotides (cGMP) role in carcinoma growth and invasivity. We analyzed two cell lines, LSHT29 and 17GT, and tissues in patients with carcinoma and malignant tissues with (N+) and without (N-) lymph node metastases. Higher cGMP levels in pathological samples suggest a strong correlation between intracellular cGMP concentration and carcinoma progression.     

Differences in islet-enriched miRNAs in healthy and glucose intolerant human subjects

Many microRNAs (miRNAs) are known to be cell-type specific and are implicated in development of diseases. We investigated the global expression pattern of miRNAs in human pancreatic islets compared to liver and skeletal muscle, using bead-based technology and quantitative RT-PCR. In addition to the known islet-specific miR-375, we also found enrichment of miR-127-3p, miR-184, miR-195 and miR-493∗ in the pancreatic islets. The expression of miR-375, miR-127-3p, miR-184 and the liver-enriched miR-122 is positively correlated to insulin biosynthesis, while the expression of miR-127-3p and miR-184 is negatively correlated to glucose-stimulated insulin secretion (GSIS). These correlations were absent in islets of glucose intolerant donors (HbA1c ? 6.1). We suggest that the presence of an islet-specific miRNA network, which consists of at least miR-375, miR-127-3p and miR-184, potentially involved in insulin secretion. Our results provide new insight into miRNA-mediated regulation of insulin secretion in healthy and glucose intolerant subjects.