Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS.     

Right Limbic FDG-PET Hypometabolism Correlates with Emotion Recognition and Attribution in Probable Behavioral Variant of Frontotemporal Dementia Patients

The behavioural variant of frontotemporal dementia (bvFTD) is a rare disease mainly affecting the social brain. FDG-PET fronto-temporal hypometabolism is a supportive feature for the diagnosis. It may also provide specific functional metabolic signatures for altered socio-emotional processing. In this study, we evaluated the emotion recognition and attribution deficits and FDG-PET cerebral metabolic patterns at the group and individual levels in a sample of sporadic bvFTD patients, exploring the cognitive-functional correlations. Seventeen probable mild bvFTD patients (10 male and 7 female; age 67.8±9.9) were administered standardized and validated version of social cognition tasks assessing the recognition of basic emotions and the attribution of emotions and intentions (i.e., Ekman 60-Faces test-Ek60F and Story-based Empathy task-SET). FDG-PET was analysed using an optimized voxel-based SPM method at the single-subject and group levels. Severe deficits of emotion recognition and processing characterized the bvFTD condition. At the group level, metabolic dysfunction in the right amygdala, temporal pole, and middle cingulate cortex was highly correlated to the emotional recognition and attribution performances. At the single-subject level, however, heterogeneous impairments of social cognition tasks emerged, and different metabolic patterns, involving limbic structures and prefrontal cortices, were also observed. The derangement of a right limbic network is associated with altered socio-emotional processing in bvFTD patients, but different hypometabolic FDG-PET patterns and heterogeneous performances on social tasks at an individual level exist.     

New mammalian cellular systems to study mutations introduced at the break site by non-homologous end-joining

The non-homologous end-joining (NHEJ) pathway is a mechanism to repair DNA double strand breaks, which can introduce mutations at repair sites. We constructed new cellular systems to specifically analyze sequence modifications occurring at the repair site. In particular, we looked for the presence of telomeric repeats at the repair junctions, since our previous work indicated that telomeric sequences could be inserted at break sites in germ-line cells during primate evolution. To induce specific DNA breaks, we used the I-SceI system of Saccharomyces cerevisiae or digestion with restriction enzymes. We isolated human and hamster cell lines containing the I-SceI target site integrated in a single chromosomal locus and we exposed the cells to a continuous expression of the I-SceI endonuclease gene. Additionally, we isolated human cell lines that expressed constitutively the I-SceI endonuclease and we introduced the target site on an episomal plasmid stably transfected into the cells. These strategies allowed us to recover repair junctions in which the I-SceI target site was modified at high frequency (100% in hamster cells and about 70% in human cells). Finally, we analyzed junctions produced on an episomal plasmid linearized by restriction enzymes. In all the systems studied, sequence analysis of individual repair junctions showed that deletions were the most frequent modifications, being present in more than 80% of the junctions. On the episomal plasmids, the average deletion length was greater than at intrachromosomal sites. Insertions of nucleotides or deletions associated with insertions were rare events. Junction organization suggested different mechanisms of formation. To check for the insertion of telomeric sequences, we screened plasmid libraries representing about 3.5 x 10(5) junctions with a telomeric repeat probe. No positive clones were detected, suggesting that the addition of telomeric sequences during double strand break repair in somatic cells in culture is either a very rare event or does not occur at all.     

Urinary electrophoretic profiles from chronic fatigue syndrome and chronic fatigue syndrome/fibromyalgia patients: a pilot study for achieving their normalization

Aim of our study was to determine if there were distinct, disease-related patterns of urinary analytes in chronic fatigue syndrome (CFS) and chronic fatigue syndrome/fibromyalgia (CFS/FM) compared to normal controls (NC). Urine was collected from these subjects for two consecutive 24 h periods and aliquots were submitted to micellar electrokinetic chromatography (MEKC). To compensate for the differences in peak migration times, these were normalized from the 35 min duration of run to a 100-point scale, and each peak was assigned its normalized time measure. Peak heights were also normalized by dividing the mAU by that of the internal standard (creatinine) and multiplying by 100. MEKC with normalization for peak height and migration time generated comparable results within each of the patient groups. CFS/FM and CFS had significant differences in peaks compared to NC that may be of significance as biomarkers of illnesses.     

Proteomic changes in rat serum, polymorphonuclear and mononuclear leukocytes after chronic nicotine administration

In order to gain information about the effect triggered at the molecular level by nicotine, its neuroimmunomodulatory properties and its impact on the pathogenesis of inflammatory diseases, peripheral blood serum and leukocytes of rat submitted to passive nicotine administration were subjected to proteomic investigation. Serum, polymorphonuclear (PMN) and mononuclear (MN) leukocytes from chronically treated animals and from control animals were analysed by a two-dimensional (2-D) gel electrophoresis/mass spectrometry approach to detect differentially expressed proteins. The nicotine regimen selected is known to have a stimulatory effect on locomotor activity and to produce a sensitisation of the mesolimbic dopamine system mechanism involved in addiction development. After 2-D gel analysis and matching, 36 spots in serum, seven in PMN and five in MN were found to display a statistical difference in their expression and were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry peptide fingerprinting for protein identification. Fifteen different proteins were identified. The results indicate an overall impact of nicotine on proteins involved in a variety of cellular and metabolic pathways, including acute phase response (suggesting the effect on inflammatory cascades and more in general on the immune system), oxidative stress metabolism and assembly and regulation of cytoskeleton. In particular, the observed changes imply a general reduction in the inflammatory response with a concomitant increased unbalance of the oxidative stress metabolism in the periphery and point to a number of potential noninvasive markers for the central nervous system (CNS) and non-CNS mediated activities of nicotine.     

Spot overlapping in two-dimensional maps: a serious problem ignored for much too long

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.     

Proteome analysis for the identification of in vivo estrogen-regulated proteins in bone

Estrogen deficiency results in a reduced bone mass, which can be prevented by treatment with estrogens. This study used a proteomic approach for the first time to obtain a global perspective of estrogens' effects on whole-bone proteins. Bone proteome profiles were examined in three groups of mice: (1) sham-operated with normal ovarian functions, (2) ovariectomised and (3) ovariectomised with estrogen replacement therapy. Bone proteins extracted from the humerus were separated by 2-DE and visualised by CBB colloidal staining. Spot detection and quantification was done by image analysis. Differentially expressed proteins were identified by MS and database search, using peptide mass fingerprint and peptide sequence analysis. Differential expression analysis in the three experimental groups showed significant changes for 14 proteins. These included proteins related to bone metabolism, cytoskeleton components and energy metabolic pathways. Our data suggest that some proteins related to cytoskeleton and to energy pathways, such as tropomyosins, aconitase 2 and enolase beta, might be new molecular targets responsive to the effects of estrogen. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the pleiotropic effects of estrogens on bone.     

Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins

The human salivary acidic proline-rich proteins (aPRPs) complex was investigated by different chromatographic and mass spectrometric approaches and the main aPRPs, namely PRP-1, PRP-2 and PIF-s (15,515 amu), Db-s (17,632 amu) and Pa (15,462 amu) proteins, were detected. All these isoforms are phosphorylated at Ser-8 and Ser-22 and have a pyroglutamic moiety at the N-terminus. Apart from Pa, all the other aPRPs undergo a proteolytic cleavage at Arg-106 residue (Arg-127 in Db-s protein), that generates the small PC peptide (4371 amu) and PRP-3, PRP-4, PIF-f (11,162 amu) and Db-f (13,280 amu) proteins, all of which were detected. With regard to the Pa protein, the main form detected was the dimeric derivative (Pa 2-mer, 30,922 amu) originated by a disulfide bond involving Cys-103 residue. Besides these known isoforms, several previously undetected aPRP derivatives were found (in minor amounts): (i) the triphosphorylated derivatives of PRP-1/PRP-2/PIF-s and Db-s, showing the additional phosphate group at Ser-17; (ii) the mono-phosphorylated forms at either Ser-22 or Ser-8 of PRP-1/PRP-2/PIF-s, PRP-3/PRP-4/PIF-f, Db-s and Db-f; (iii) a nonphosphorylated form of PRP-3/PRP-4/PIF-f; (iv) the triphosphorylated and diphosphorylated forms of Pa 2-mer. Moreover, minor quantities of PRP-3/PRP-4/PIF-f lacking the C-terminal Arg (11,006 amu), and of Pa 2-mer lacking the C-terminal Gln (30,793 amu) were found. By this approach the different phenotypes of PRH1 locus in 59 different subjects were characterized.