A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis

Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype–phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype–phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype–phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway.     

Serum miR-30c-5p is a potential biomarker for multiple system atrophy

Multiple system atrophy (MSA) is a neurodegenerative disease that belongs to the α synucleinopathies. Clinically, there is an overlap between MSA and Parkinson’s disease (PD), especially at the early disease stage. However, these two pathologies differ in terms of disease progression. Currently, no biomarker exists to differentiate MSA from PD. MicroRNAs are non-coding RNAs implicated in gene expression regulation. MiRNAs modulate cellular activity and they control a range of physiological and pathological functions. miRNAs are found in biofluids, such as blood, serum, plasma, saliva, and cerebrospinal fluid. Many groups, including ours, found that circulating miRNAs are differently expressed in blood, plasma, serum and cerebrospinal fluid of PD and MSA patients. In the present study, our primary aim was to determine if serum mir-30-5p and mir-148b-5p can be used as biomarkers for early diagnosis of PD and/or MSA. Our secondary goal was to determine if serum levels of those miRNAs can be correlated with the patients’ clinical profile. Using quantitative PCR (qPCR), we evaluated expression levels of miR-30c-5p and miR148b-5p in serum samples from PD (n?=?56), MSA (n?=?49), and healthy control (n?=?50) subjects. We have found that miR-30c-5p is significantly upregulated in MSA if compared with PD and healthy control subjects. Moreover, serum miR-30c-5p levels correlate with disease duration in both MSA and PD. No significant difference was found in miR-148b-5p among MSA, PD and healthy control subjects. Our results suggest a possible role of serum miR-30-5p as a biomarker for diagnosis and progression of MSA.

     

Simultaneous reversed-phase high-performance liquid chromatographic separation of non-polar isoprenoid lipids and their determination

A procedure for the rapid identification and determination of non-polar isoprenoid lipids from animal tissues was developed. The complete determination can be carried out by reversed-phase HPLC of just two samples. The first, extracted from unaltered tissues and suitably processed by column chromatography, provides information about free cholesterol, cholesteryl esters, coenzymes Q, free dolichols and dolichyl esters. The second, obtained from saponified tissues, can be used to detect both total cholesterol and total dolichols. Specific calibration graphs were constructed for the determination of the different constituents.     

Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants

Metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, has been a target for antineoplastic therapy since these naturally occurring alkyl amines were found essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. A correlation between regulation of cell proliferation and polyamine metabolism is described. In particular, polyamine catabolism involves copper-containing amine oxidases and FAD-dependent polyamine oxidases. Several studies showed an important role of these enzymes in several developmental and disease-related processes in both animals and plants through a control on polyamine homeostasis in response to normal cellular signals, drug treatment, environmental and/or cellular stressors. The production of toxic aldehydes and reactive oxygen species, H(2)O(2) in particular, by these oxidases using extracellular and intracellular polyamines as substrates, suggests a mechanism by which the oxidases can be exploited as antineoplastic drug targets. This minireview summarizes recent advances on the physiological roles of polyamine catabolism in animals and plants in an attempt to highlight differences and similarities that may contribute to determine in detail the underlined mechanisms involved. This information could be useful in evaluating the possibility of this metabolic pathway as a target for new antiproliferative therapies in animals and stress tolerance strategies in plants.     

Effect of peroxides on spermine transport in rat brain and liver mitochondria

The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs.     

Evidence for pH-dependent multiple conformers in iron(II) heme–human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.