In vitro functional characterization of missense mutations in the LDLR gene.

BACKGROUND: Mutations in the LDL receptor gene are the major cause of familial hypercholesterolaemia (FH) but it has been previously shown that the simple finding of a variation in the coding sequence of the LDLR does not confirm that it is the actual cause of FH. The pathogenicity of five missense alterations in the LDLR gene coding sequence found in a previous epidemiologic study was investigated. METHODS: The effects of the different sequence variants on LDLR expression and activity were analysed in vitro stably transfected CHO-ldlA7 cells by immunobloting of cell extracts, by uptake and degradation rates of (125)I-labelled LDL and immunofluorescence microscopy of whole cells. Analysis in silico was also performed. RESULTS: LDLR functional assays showed that variants p.V429L, p.W490R and p.S648P of the LDLR coding sequence severely impaired receptor function, while variant p.P685S had a milder effect and cells carrying p.V859M variant had LDL clearance rates comparable to cells expressing normal LDLR. In silico analysis failed to predict correctly the effect of 4/5 alterations. CONCLUSION: Assessing the pathogenicity of the different variants found in patients with clinical diagnosis of FH is of great importance to distinguish pathogenic mutations from rare silent variants and has clinical implications for determining the associated cardiovascular risk.

cAMP acts as a second messenger in pollen tube growth and reorientation.

Pollen tube growth and reorientation is a prerequisite for fertilization and seed formation. Here we report imaging of cAMP distribution in living pollen tubes microinjected with the protein kinase A-derived fluorosensor. Growing tubes revealed a uniform distribution of cAMP with a resting concentration of approximately 100-150 nM. Modulators of adenylyl cyclase (AC), forskolin, and dideoxyadenosine could alter these values. Transient elevations in the apical region could be correlated with changes in the tube-growth axis, suggesting a role for cAMP in polarized growth. Changes in cAMP arise through the activity of a putative AC identified in pollen. This signaling protein shows homology to functional motifs in fungal AC. Expression of the cDNA in Escherichia coli resulted in cAMP increase and complemented a catabolic defect in the fermentation of carbohydrates caused by the absence of cAMP in a cyaA mutant. Antisense assays performed with oligodeoxynucleotide probes directed against conserved motifs perturbed tip growth, suggesting that modulation of cAMP concentration is vital for tip growth.

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells.

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.