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Chloride conductance in membrane vesicles from human placenta using a fluorescent probe. Implications for cystic fibrosis
No full textPrevious evidence suggests that the molecular defect in cystic fibrosis (CF) could reside in an altered chloride conductance of epithelial tissues. Since the brush border of the syncytiotrophoblast of the chorionic villi of human placenta is an abundant source of epithelial membranes and it is unaltered by secondary pathology or treatment we chose to characterize its chloride conductance and to compare it in normal and CF membranes. Chloride transport was studied in microvillar vesicles (MVV) by the quencing of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Chloride conductance at 23°C: (a) increased by 39% under a membrane potential change of 70 mV; (b) was inhibited by diphenylamine 2-carboxylate (K(i) = 150 μM); (c) displayed an activation energy of 3.5 kcal · mol-1. The comparison of the chloride conductance for an inwardly directed gradient of 150 mM Cl- at 23°C (membrane potential set at 0 mV) between CF and control membranes was not significantly different. These findings demonstrate the presence of a chloride conductive pathway in microvillar vesicles from human placenta and preliminary results exclude major differences in the conductance of CF derived material in the absence of neurohormonal stimuli
Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene
No full textThe regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator (CFTR) is phosphorylated by protein kinase A and protein kinase C (PKC) in vivo (Picciotto, M. R., Cohn, J. A., Bertuzzi, G., Greengard, P., and Nairn, A. C. (1992) J. Biol. Chem. 267, 12742-12752), but so far the functional effect of the PKC-dependent phosphorylation has not been clarified. We investigated the effect of PKC on the CFTR-mediated Cl- transport by treating with phorbol 12-myristate 13-acetate (PMA), the cell line C127i stably expressing CFTR wild type (C127 CFTRw/t), or CFTR bearing the most common mutation deltaF508 (C127 CFTRdF508). We show that PMA activates Cl- efflux in C127 CFTRw/t, but not in C127 CFTRdF508 and C127i. The PMA-dependent activation of CFTR is not mediated by increase of intracellular [cAMP] and is not the result of a primary activation of a K+ conductive pathway. These results strongly suggest that PKC activates directly CFTR-mediated Cl- transport
Heparan Sulfate Glycosaminoglycans Are Involved in Adenovirus Type 5 and 2-Host Cell Interactions
No full textAbstractGene therapy vectors derived from subgroup C adenoviruses of the serotype 5 (Ad5) and 2 (Ad2) resulted in inefficient infection of well differentiated respiratory cells, both in vitro and in vivo. The level of expression and localization of the primary receptor for Ad5 and Ad2, termed CAR, do not completely explain why the infection efficiency varies greatly in different experimental conditions. The possibility that additional receptors like proteoglycans are involved in the infection of Ad5 and Ad2 was investigated, because several pathogenic microorganisms use heparan sulfate–glycosaminoglycans (HS-GAGs) as coreceptors for multistep attachment to target cells. The HS-GAG analog heparin decreased Ad5- and Ad2-mediated infection and binding starting from the concentration of 0.1 μg/ml, up to a maximum of 50%. A similar reduction in Ad5 binding and infection was obtained by treatment of cells with heparin lyases I, II, and III but not with chondroitin ABC lyase. The effect of heparin on Ad5 binding has not been observed in surface GAG-defective Raji cells and after treating A549 cells with heparin lyases I, II ,and III. The binding of Ad5 was completely abolished when both CAR was blocked with RmcB antibody and HS-GAGs were competitively inhibited by heparin. Parallel experiments demonstrate that HS-GAGs are irrelevant to binding and infection of the subgroup B adenovirus type 3. Collectively, these results demonstrate for the first time that HS-GAGs expressed on the cell surface are involved in the binding of Ad5 and Ad2 to host cells
Circulating microRNAs as emerging non-invasive biomarkers for gliomas
No full textNo single circulating biomarker has been put to practice for malignant gliomas so far, the most lethal primary brain tumors. Many promising protein biomarkers such as the mutant EGFRvIII or glial fibrillary acidic protein (GFAP) have already been detected in the blood and cerebrospinal fluid (CSF) of patients with gliomas, but their clinical value is still pending validation. Furthermore, these and other proteins seem to lack sufficient sensitivity and specificity required for a successful biomarker in this clinical setting. The expression profiling of microRNAs (miRNAs) has already entered cancer clinics as diagnostic and prognostic biomarkers, for assessing tumor initiation, progression and response to treatment. Large-scale miRNA expression analyses reported both up-regulation and down-regulation of several miRNAs in tumour tissues from patients with gliomas compared to normal brain tissue, thus supporting the development of miRNA-based biomarkers. Using comprehensive high-throughput approaches, such as microarrays, different circulating miRNAs were proposed as potential biomarkers of gliomas. This review is aimed to summarize the clinical evidence about circulating miRNA biomarkers discovered to date. Mandatory issues to develop clinically validated biomarkers to improve time of diagnosis, predicting response to treatment and prognosis of patients with gliomas are also herein addresses
IMINOSUGAR-BASED INHIBITORS OF CERAMIDE GLUCOSYL-TRANSFERASE (GLCCERT) AND NONLYSOSOMAL GLUCOSYLCERAMIDASE (GBA2) REDUCE THE INFLAMMATORY RESPONSE TO P. AERUGINOSA IN CF BRONCHIAL EPITHELIAL CELLS
No full textNo availabl
Silencing of genes coding for transcription factors: biological effects of decoy oligonucleotides on cystic fibrosis bronchial epithelial cells
No full textChronic pulmonary inflammation in the lungs of patients affected by cystic fibrosis is characterized by massive intra-bronchial recruitment of neutrophils, initiated and sustained upon interaction of pathogens (including Pseudomonas aeruginosa) with surface bronchial cells, associated with release of chemokines, including interleukins IL-8 and IL-6. This process leads to progressive tissue damage. In order to develop novel therapeutic strategies altering this process, the transcription factor (TF) decoy approach was applied, based on the intracellular delivery of double stranded oligodeoxynucleotides (ODNs) mimicking the binding sites of transcription factors and causing inhibition of the binding of TF-related proteins to regulatory sequences present in the promoter of specific genes. Since the promoters IL-8 and IL-6 contain consensus sequences for NF-kB and Sp1, IB3-1 cells were transfected with double stranded TF “decoy” ODNs targeting NF-kB and Sp1. Inhibition of Sp1 activity using transcription factor decoy molecules leads to inhibition of the expression of IL-6 gene; in addition, NF-kB decoy ODNs produced inhibition of transcription of IL-8. In conclusion, these results suggest that intracellular delivery of “decoy” molecules aimed to compete with the NF-kB and Sp1 transcription factors is a promising strategy to obtain selective inhibition of expression of IL-8 and IL-6 genes stimulated in epithelial cells of the respiratory tract upon surface interaction with bacteria
Heparan Sulfate Glycosaminoglycans Are Receptors Sufficient To Mediate the Initial Binding of Adenovirus Types 2 and 5
No full textABSTRACT
Cell infection by adenovirus serotypes 2 and 5 (Ad2/5) initiates with the attachment of Ad fiber to the coxsackievirus and Ad receptor (CAR) followed by α
v
integrin-mediated entry. We recently demonstrated that heparan sulfate glycosaminoglycans (HS GAGs) expressed on cell surfaces are involved in the binding and infection of Ad2/5 (M. C. Dechecchi, A. Tamanini, A. Bonizzato, and G. Cabrini, Virology 268:382–390, 2000). The role of HS GAGs was investigated using extracellular soluble domain 1 of CAR (sCAR-D1) and heparin as soluble receptor analogues of CAR and HS GAGs in A549 and recombinant CHO cell lines with differential levels of expression of the two receptors and cultured to various densities. Complete inhibition of binding and infection was obtained by preincubating Ad2/5 with both heparin (10 μg/ml) and sCAR-D1 (200 μg/ml) in A549 cells. Partial inhibition was observed when heparin and sCAR-D1 were preincubated separately with Ad. The level of heparin-sensitive [
3
H]Ad2/5 binding doubled in sparse A549 cells (50 to 70,000 cells/cm
2
) with respect to that of cells grown to confluence (200 to 300,000 cells/cm
2
), in parallel with increased expression of HS GAGs. [
3
H]Ad2 bound to sparse CAR-negative CHO cells expressing HS GAGs (CHO K1). No [
3
H]Ad2 binding was observed in CHO K1 cells upon competitive inhibition with heparin and in HS GAG-defective CHO A745, D677, and E606 clones. HS-sensitive Ad2 infection was obtained in CAR-negative sparse CHO K1 cells but not in CHO A745 cells, which were permissive to infection only upon transfection with CAR. These results demonstrate that HS GAGs are sufficient to mediate the initial binding of Ad2/5.
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Modulators of Sphingolipid Metabolism Reduce Lung Inflammation
No full textInvestigation on novel targets for the treatment of Cystic Fibrosis (CF) lung inflammation is a major priority, considering that no effective therapy is available for this purpose. Consistently with the evidence that the sphingolipid (SL) ceramide regulates airway inflammation and infection in CF mice and patients, SLs have been identified as targets to treat pulmonary disorders, including CF. Because miglustat, an inhibitor of the synthesis of glycosphingolipids, reduces the P.aeruginosa-dependent transcription of IL-8 gene in bronchial cells, we examined the effect of miglustat and amitriptyline, another drug affecting ceramide metabolism, on expression of 92 genes implicated in host immune defence. Infection with P.aeruginosa strain PAO1 up-modulated the expression of 14 (27%) genes in IB3-1 cells and 15 (29%) in CF primary respiratory epithelium grown at air-liquid interface, including chemokines (IL-8, Gro-α/β/γ, GCP-2), pro-inflammatory citokines (IL-1α/β, IL-6, TNF-α, ICAM-1, NFKB1, TLR-2 and HBD-4 genes, confirming that bronchial epithelium is an important source of inflammatory mediators. Both miglustat and amitriptyline reduced the immune response, an effect that went in parallel with a decrease in P.aeruginosa-induced ceramide accumulation. Miglustat (100mg/Kg), given to C57BL/6 mice once daily for a period of three consecutive days before lipopolysaccharide (LPS) challenge, strongly reduced the amount of neutrophils recruited in the airways and the expression of the chemokine KC in lung extracts. Collectively these results indicate that targeting SLs metabolism can down-modulate the recruitment of neutrophils into the lung
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