Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in [³H]mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [³H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 58 000}$), there are several minor surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 76 000, 86 000}\text{and}\text{92 000–100 000}$) which are apparent extrinsic membrane components, and two surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 42 000}\text{and}\text{130 000}$) which are intrinsic membrane components.     

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells

Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.     

Cortactin activation by FVIIa/tissue factor and PAR2 promotes endothelial cell migration

Cellular migration is a complex process that requires the polymerization of actin filaments to drive cellular extension. Smooth muscle and cancer cell migration has been shown to be affected by coagulation factors, notably the factor VII (FVIIa) and tissue factor (TF) complex. The present studies delineated mediators involved with the process of FVIIa/TF-induced cell migration and utilized a simple, precise, and reproducible, migration assay. Both FVIIa and protease-activated receptor-2 (PAR2)-activating peptide, SLIGRL, increased the migration rate of porcine cerebral microvascular endothelial cells (pCMVECs) overexpressing human TF. Ras homolog gene family member A (RhoA) and cortactin were upregulated during the process; expression of HIF, actin polymerization nuclear diaphanous-related formin-1 and -2 (Dia1, and Dia2) were unaffected. Gene silencing by shRNA to PAR2, RhoA, and cortactin attenuated this gene upregulation and migration induced by FVIIa/TF. Utilizing immunocellular localization, we demonstrate that during FVIIa/TF and PAR2 activation, cortactin molecules translocate from the cytoplasm to the cell periphery and assist in lamellipodia formation of pCMVECs. Overall, we demonstrate a novel regulation and role for cortactin in FVIIa/TF-mediated endothelial cell migration that occurs through a PAR2 and RhoA dependent mechanism.     

Interaction between bacterial enteric pathogens and aquatic macrophytes. Can Salmonella be internalized in the plants used in phytoremediation processes?

Abstract

The environment is considered a reservoir of pathogens and a possible source of infection for animals and humans. The association between enteric pathogens and food plants has been demonstrated in several studies, while few studies have addressed possible interactions between human pathogens and aquatic plants. This study, performed by setting mesocosms, evaluates the interaction between an enteric pathogen (Salmonella enterica serovar Napoli, S. Napoli) and a macrophyte (Phragmites australis (Cav.) Trin. ex Steudel) and the possible ability of the bacterium to internalize into the plant. The results show that S. Napoli concentration decreased gradually in growth solution without plants (control) while it was able to persist adhering to submerged parts of plants in treated mesocosms. The adhesion of the bacterium remained stable for 20?days, then decreased gradually until the end of the experiment. In addition, S. Napoli was able to internalize and colonize stems and leaves. In conclusion, the study suggests that macrophytes can represent an alternative environmental reservoir of pathogens for humans and animals. The adhesion to roots and rhizomes and the internalization could contribute to the bacterial persistence in the aquatic ecosystems by playing an important role in ecology and transmission of pathogens.     

Supervised learning methods for the recognition of melanoma cell lines through the analysis of their Raman spectra

Malignant melanoma is an aggressive form of skin cancer, which develops from the genetic mutations of melanocytes – the most frequent involving BRAF and NRAS genes. The choice and the effectiveness of the therapeutic approach depend on tumour mutation; therefore, its assessment is of paramount importance. Current methods for mutation analysis are destructive and take a long time; instead, Raman spectroscopy could provide a fast, label-free and non-destructive alternative. In this study, confocal Raman microscopy has been used for examining three in vitro melanoma cell lines, harbouring different molecular profiles and, in particular, specific BRAF and NRAS driver mutations. The molecular information obtained from Raman spectra has served for developing two alternative classification algorithms based on linear discriminant analysis and artificial neural network. Both methods provide high accuracy (≥90%) in discriminating all cell types, suggesting that Raman spectroscopy may be an effective tool for detecting molecular differences between melanoma mutations.