Effects of positive airway pressure therapy on cardiovascular and metabolic markers in males with obstructive sleep apnea.

INTRODUCTION: Obstructive sleep apnea syndrome (OSAS) is associated with cardiovascular/metabolic complications. Some analytical parameters (homocysteine, glycemic and lipidic profiles) are recognized markers of these consequences. Limited data is available on the association of these markers and OSAS's severity/response to positive airway pressure therapy (PAP). MATERIAL AND METHODS: In this prospective study we analyzed polysomnographic and analytical data of male patients admitted to sleep laboratory. The aim was to evaluate metabolic/cardiovascular markers in snorers and OSAS patients, to relate with sleep parameters and PAP response. One-hundred and three patients were included, and 73 (71%) were OSAS patients. OSAS patients were similar to snorers except for higher body mass index (BMI) and dyslipidemia. Severe OSAS patients showed higher glycemia, HbA1c, insulin, and insulin resistance, and lower HDL cholesterol in comparison to mild-moderate (p<0.05, p<0.05, p<0.001, p<0.001, p<0.05, respectively). Glycemic profile and triglycerides were slightly correlated with OSAS severity. 46 OSAS patients were submitted to 6 months of PAP, with a statistical decrease in mean values of homocysteine, glycemia, total and LDL cholesterol (p<0.05, p<0.05, p<0.05, respectively), and in glycemia and LDL cholesterol in severe group only (p<0.05, p<0.05, respectively). RESULTS: This study demonstrated an association between glucose metabolism parameters and triglycerides with OSAS severity underlying the complexity of the process leading to cardiovascular/metabolic complications in this disorder. Moreover, homocysteine, glycemic and lipidic profiles changed significantly after 6 months of PAP therapy in OSAS, supporting its cardiovascular and metabolic protective effect. CONCLUSION: Our study has reinforced the importance of analytical cardiovascular/metabolic evaluation as complementary tool of diagnosis/treatment response in OSAS.

Hematological evaluation in males with obstructive sleep apnea before and after positive airway pressure.

Obstructive sleep apnea syndrome (OSAS) is a systemic inflammatory disease associated with cardiovascular consequences. Red blood cell distribution width (RDW), mean platelet volume (MPV), and platelet distribution width (PDW) are recognized biomarkers of cardiovascular morbidity/mortality. Limited data is available on the association between these parameters and OSAS severity and the relationship with positive airway pressure therapy (PAP). In this prospective study of male OSAS patients we analyzed hematological data in order to evaluate their value in predicting OSAS severity, the relationship with sleep parameters, and their behavior under PAP. Seventy-three patients were included (mean age 46.5 years), of which 36 were mild (49.3%), 10 moderate (13.7%), and 27 severe (37%). The mean RDW increased significantly with OSAS severity and showed a positive correlation with respiratory disturbance index and hypoxemic burdens. Additionally, a group of 48 patients (mean age 47.2 years) were submitted to PAP. After six months, red blood cell count, hemoglobin, hematocrit, and platelet count showed a significant decrease (p<0.0001; p<0.0001; p=0.001; p<0.0001; respectively). Concerning OSAS severity, these parameters also significantly decreased in mild patients (p=0.003; p=0.043; p=0.020; p=0.014; respectively) but only hemoglobin, hematocrit, and platelet count decreased in severe cases (p<0.0001; p=0.008; p=0.018; respectively). This study demonstrated an association between RDW values and OSAS severity. Moreover, red cell and platelet parameters changed significantly after PAP, supporting its cardiovascular protective effect. RDW may become a simple/inexpensive blood biomarker, making it useful in prioritizing OSAS patients waiting for polysomnography, and red cell and platelet parameters could be useful in PAP follow up.

Clinical proteomics stretch goals: EuPA 2012 roundtable report.

The field of clinical proteomics is faced with multiple challenges which need to be overcome in order to improve our understanding of human diseases and provide management solutions. Researchers interested in clinical proteomics assembled for a roundtable discussion at the European Association for Proteomics (EuPA) conference held in Glasgow in July 2012, to discuss these challenges and highlight the key areas for successful clinical proteomic studies. This report shares topics of discussion and the resulting stretch goals of clinical proteomics for researchers to strive towards.

EuPA achieves visibility - an activity report on the first three years.

Plans for the European Proteomics Association (EuPA) were conceived and established during 2004 and 2005, and culminated in the formal inception of the organisation during the 4th HUPO World Congress held in Munich in 2005. The mission from the outset has been three-tiered and is to: i) strengthen the national Proteomics organizations in their efforts; ii) to co-ordinate and provide educational programs, and iii) to advance the networking of scientists through meetings, workshops and student exchange. Linked to the mission were objectives to emphasise the benefits and contributions of Proteomics to biological and industrial researchers, the general public and science policy makers in Europe. In addition, the EuPA set out to promote scientific exchange for all applications and technology development related to Proteomics, and coordinate joint activities of national Proteomics societies at the European level. To achieve these tasks an organisational structure was conceived whereby four Activity Committees (Conferences/Communications, Education, EuPA-HUPO-Interactions and Funding) were implemented and a General Council consisting of all member countries. The remarkable rise and progress the EuPA has achieved in this small time frame is reported here.

Ionic transport in tall columnar epithelial (TCE) cells obtained by nasal brushing from non-cystic fibrosis (CF) individuals.

Tall columnar epithelial (TCE) cells can be obtained by a non-invasive procedure through brushing the inferior turbinate and the adjacent lateral nasal wall. Here, we present results from the functional study of epithelial cells, thus obtained by using the patch-clamp technique. By patch-clamping the sub-apical region of TCE cells, we were able to identify at least three different groups of Cl- channels, namely: a) one with large conductance, rectifying, which was the most frequently found type of Cl- channel; b) a second type of small conductance, activated by cAMP and IBMX, in excised inside-out patches and voltage independent; c) a third type with a conductance around 25 pS, voltage independent, with a linear IV relationship, that could be observed in the excised inside-out configuration. The study of CFTR Cl- channel and its role in airway cell physiology has generally been conducted in cultured cells, most of which not polarized. This experimental work using freshly obtained TCE cells from the nasal epithelium, demonstrates that such cells may be one valid tool to study Cl- channels (most probably ORCC and CFTR Cl- channels) as a model for the lower respiratory epithelium.

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.

Cystic fibrosis patients with the 3272-26A-->G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane.

We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.

A Tetrahymena orthologue of the mouse chaperonin subunit CCT gamma and its coexpression with tubulin during cilia recovery.

A Tetrahymena pyriformis gene orthologue to the mouse CCT gamma gene has been cloned and sequenced. The coding region of this gene, named TpCCT gamma, is interrupted by six introns and codes for a protein of 559 amino acid residues. This protein shares a 57.6% identity with the mouse CCT gamma protein whereas in average a 30% identity was found with CCT alpha proteins from mammalian to yeast. The expression of this gene was investigated by Northern blot hybridization in Tetrahymena exponentially growing cells and cells during cilia recovery. The TpCCT gamma gene is expressed producing a unique transcript of 2.1 kilobases. A relevant aspect of our findings is the coordinated response of TpCCT gamma and tubulin genes to cilia regeneration. TpCCT gamma and tubulin transcripts reached higher levels between 45-120 min of reciliation compared to those found in exponentially growing cells. These high levels could be the result of the increase in apparent transcription rates detected at 60 min of reciliation. We also observed that TpCCT gamma transcripts are not increased after exposure of Tetrahymena cells to a heat-shock (28-->34 degrees C). Our results seem to indicate an important role of this gene in the differentiation process of ciliogenesis.

Multiple alpha-tubulin isoforms in cilia and cytoskeleton of Tetrahymena pyriformis generated by post-translational modifications. Studies during reciliation.

alpha-Tubulin microheterogeneity was studied in Tetrahymena pyriformis. Using two-dimensional electrophoretic analysis, we found between five and seven alpha-tubulin and four beta-tubulin isoforms in cilia and four or five alpha-tubulins and two beta-tubulins in cytoskeleton. Immunoblotting assay with anti-(acetyl alpha-tubulin) monoclonal antibody 6-11B-1 and [3H]acetate labelling revealed that the alpha-tubulin isoforms are post-translationally modified by acetylation. Our results also show that tubulins in the soluble cytoplasmic fraction are not acetylated. Nevertheless, a slight reaction with the antibody 6-11 B-1 can be observed in the taxol and vinblastine-treated cytoplasmic pool. Pulse/chase experiments using [35S]methionine during cell reciliation have demonstrated that the basic alpha-tubulin isoforms are converted into acidic isoforms in the absence of protein synthesis, suggesting that the basic alpha-tubulin is the precursor of the acidic forms which are found in cilia and cytoskeleton. In-vivo-translation selection demonstrated the existence of a single precursor molecule which corresponded to the most basic alpha-tubulin. Taken together, our results provide evidence for the existence of post-translational modifications, namely acetylation. Nevertheless, other post-translational mechanisms involved in the biosynthesis of microtubules of cilia and cytoskeleton are required to explain the whole alpha-tubulin heterogeneity.

Sequence of one alpha- and two beta-tubulin genes of Tetrahymena pyriformis. Structural and functional relationships with other eukaryotic tubulin genes.

Macronuclear DNA of the ciliate Tetrahymena pyriformis contains only one size class of fragments coding for alpha-tubulin, alpha TT. We have isolated alpha TT from a partial plasmid library, using Chlamydomonas reinhardtii alpha-tubulin gene as a probe. This gene as well as the two beta-tubulin genes, beta TT1 and beta TT2, have been sequenced. None of these genes contains introns and all use TGA as the stop codon. In the coding region of the two beta-tubulin genes, there are several TAA and TAG stop codons that probably code for glutamine. The codon usage is very biased. Regions flanking the tubulin coding sequences are A + T-rich (75%) and quite different among themselves. In these regions there are several putative transcription-regulatory sequences. Nuclear transcripts begin and terminate at multiple sites. The beta-tubulin proteins differ only in two amino acid residues. Primary structure of Tetrahymena tubulins as well as their hydropathy indexes show a high degree of homology with tubulins from other organisms. Two-dimensional electrophoretic analysis of the ciliary tubulins shows the presence of eight alpha-tubulins and four beta-tubulins. The alpha-tubulins migrate faster than the beta-tubulins, in contrast with what happens with brain tubulins. We suggest that there are several alpha- and beta-tubulin isoforms and the migratory inversion observed may be due to post-translational modifications.