A comparative MudPIT analysis identifies different expression profiles in heart compartments

Cardiomyopathies indistinctly affect atrial and ventricular cardiac compartments with alterations of their mechanical and/or electrical activity. To understand the main mechanisms involved in these pathological alterations, a detailed knowledge of the physiology of the healthy heart is critical. In the present work, we utilize multidimensional protein identification technology to characterize the murine left ventricle (LV), right ventricle (RV), and atria (A) proteomes, identifying thousands of distinct proteins. Moreover, using multidimensional algorithm protein map tool, relative abundances of proteins among the heart chambers were investigated. In sum, we found 16 and 55 proteins were more abundant in LV compared to RV and A, respectively; 47 and 60 proteins were more abundant in RV than LV and A, respectively; and, 81 and 74 proteins were more abundant in A than LV and RV, respectively. This detailed characterization of myocardial compartment proteome represents an important advancement in the knowledge of heart physiology, and may contribute to the identification of key features underlying the onset of cardiomyopathy.     

The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected

Based on pre-DNA racial/color methodology, clinical and pharmacological trials have traditionally considered the different geographical regions of Brazil as being very heterogeneous. We wished to ascertain how such diversity of regional color categories correlated with ancestry. Using a panel of 40 validated ancestry-informative insertion-deletion DNA polymorphisms we estimated individually the European, African and Amerindian ancestry components of 934 self-categorized White, Brown or Black Brazilians from the four most populous regions of the Country. We unraveled great ancestral diversity between and within the different regions. Especially, color categories in the northern part of Brazil diverged significantly in their ancestry proportions from their counterparts in the southern part of the Country, indicating that diverse regional semantics were being used in the self-classification as White, Brown or Black. To circumvent these regional subjective differences in color perception, we estimated the general ancestry proportions of each of the four regions in a form independent of color considerations. For that, we multiplied the proportions of a given ancestry in a given color category by the official census information about the proportion of that color category in the specific region, to arrive at a "total ancestry" estimate. Once such a calculation was performed, there emerged a much higher level of uniformity than previously expected. In all regions studied, the European ancestry was predominant, with proportions ranging from 60.6% in the Northeast to 77.7% in the South. We propose that the immigration of six million Europeans to Brazil in the 19th and 20th centuries--a phenomenon described and intended as the "whitening of Brazil"--is in large part responsible for dissipating previous ancestry dissimilarities that reflected region-specific population histories. These findings, of both clinical and sociological importance for Brazil, should also be relevant to other countries with ancestrally admixed populations.     

Retrospective investigation of an influenza A/H1N1pdm outbreak in an Italian military ship cruising in the Mediterranean Sea, May-September 2009

Background

Clinical surveillance may have underestimated the real extent of the spread of the new strain of influenza A/H1N1, which surfaced in April 2009 originating the first influenza pandemic of the 21^st century. Here we report a serological investigation on an influenza A/H1N1pdm outbreak in an Italian military ship while cruising in the Mediterranean Sea (May 24-September 6, 2009).

Methods

The contemporary presence of HAI and CF antibodies was used to retrospectively estimate the extent of influenza A/H1N1pdm spread across the crew members (median age: 29 years).

Findings

During the cruise, 2 crew members fulfilled the surveillance case definition for influenza, but only one was laboratory confirmed by influenza A/H1N1pdm-specific RT-PCR; 52 reported acute respiratory illness (ARI) episodes, and 183 reported no ARI episodes. Overall, among the 211 crew member for whom a valid serological result was available, 39.3% tested seropositive for influenza A/H1N1pdm. The proportion of seropositives was significantly associated with more crowded living quarters and tended to be higher in those aged <40 and in those reporting ARI or suspected/confirmed influenza A/H1N1pdm compared to the asymptomatic individuals. No association was found with previous seasonal influenza vaccination.

Conclusions

These findings underline the risk for rapid spread of novel strains of influenza A in confined environment, such as military ships, where crowding, rigorous working environment, physiologic stress occur. The high proportion of asymptomatic infections in this ship-borne outbreak supports the concept that serological surveillance in such semi-closed communities is essential to appreciate the real extent of influenza A/H1N1pdm spread and can constitute, since the early stage of a pandemic, an useful model to predict the public health impact of pandemic influenza and to establish proportionate and effective countermeasures.     

BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.     

Plasma protein carbonylation and physical exercise

Regular physical activity is associated with a reduced risk of coronary heart disease, as it probably modifies the balance between free-radical generation and antioxidant activity. On the other hand, however, acute physical activity increases oxygen uptake and leads to a temporary imbalance between the production of reactive oxygen and nitrogen species (RONS) and their disposal: this phenomenon is called oxidative stress. Proteins are one of the most important oxidation targets during physical exercise and carbonylation is one of the most common oxidative protein modifications. In cells there is a physiological level of oxidized proteins that doesn't interfere with cell function; however, an increase in oxidized protein levels may cause a series of cellular malfunctions that could lead to a disease state. For this reason the quantification of protein oxidation is important to distinguish a healthy state from a disease state. Several studies have demonstrated an increase of carbonylated plasma proteins in athletes after exercise, but none have identified targets of this oxidation. Recently a process of protein decarbonylation has been discovered, this may indicate that carbonylation could be involved in signal transduction. The aim of our research was to characterize plasma protein carbonylation in response to physical exercise in trained male endurance athletes. We analyzed by proteomic approach their plasma proteins at resting condition and after two different kinds of physical exercise (PE). We used 2D-GE followed by western blot with specific antibodies against carbonylated proteins. The 2D analysis identified Haptoglobin as potential protein target of carbonylation after PE. We also identified Serotransferrin and Fibrinogen whose carbonylation is reduced after exercise. These methods have allowed us to obtain an overview of plasma protein oxidation after physical exercise.     

Different factors affecting human ANP amyloid aggregation and their implications in congestive heart failure

Aims

Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis.

Methods and Results

A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric β-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing β-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA.

Conclusions

We established a link between IAA and CHF by ex vivo examination and assessed that β-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that β-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis.     

ALMS1-deficient fibroblasts over-express extra-cellular matrix components, display cell cycle delay and are resistant to apoptosis

Alström Syndrome (ALMS) is a rare genetic disorder (483 living cases), characterized by many clinical manifestations, including blindness, obesity, type 2 diabetes and cardiomyopathy. ALMS is caused by mutations in the ALMS1 gene, encoding for a large protein with implicated roles in ciliary function, cellular quiescence and intracellular transport. Patients with ALMS have extensive fibrosis in nearly all tissues resulting in a progressive organ failure which is often the ultimate cause of death. To focus on the role of ALMS1 mutations in the generation and maintenance of this pathological fibrosis, we performed gene expression analysis, ultrastructural characterization and functional assays in 4 dermal fibroblast cultures from ALMS patients. Using a genome-wide gene expression analysis we found alterations in genes belonging to specific categories (cell cycle, extracellular matrix (ECM) and fibrosis, cellular architecture/motility and apoptosis). ALMS fibroblasts display cytoskeleton abnormalities and migration impairment, up-regulate the expression and production of collagens and despite the increase in the cell cycle length are more resistant to apoptosis. Therefore ALMS1-deficient fibroblasts showed a constitutively activated myofibroblast phenotype even if they do not derive from a fibrotic lesion. Our results support a genetic basis for the fibrosis observed in ALMS and show that both an excessive ECM production and a failure to eliminate myofibroblasts are key mechanisms. Furthermore, our findings suggest new roles for ALMS1 in both intra- and extra-cellular events which are essential not only for the normal cellular function but also for cell-cell and ECM-cell interactions.     

Structuring detergents for extracting and stabilizing functional membrane proteins

Background

Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.

Methodology/Principal Findings

Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by ¹H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.

Conclusion/Significance

These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.     

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.