Evolutionary history of chromosome 10 in primates

We have tracked the evolutionary history of chromosomes homologous to HSA10 (PHYL-10) in primates using appropriate panels of PCP, YAC, and BAC probes. This approach allowed us to delineate more precisely the PHYL-10 constitution in the ancestor of catarrhine, platyrrhine, and prosimians. The results suggest that (i) in the ancestor of prosimians PHYL-10 was organized in two separate PHYL-10p and PHYL-10q chromosomes; (ii) in the progenitor of New World monkeys PHYL-10p was a separate chromosome, while PHYL-10q was associated with a chromosome homologous to HSA16; (iii) in the ancestor of Old World monkeys PHYL-10 was a unique chromosome with a marker order corresponding to the orang form. We have also analyzed the cat, chosen as an outgroup for its very conserved karyotype. In agreement with published data our experiments show that the PHYL-10 in cat is structured in two blocks, PHYL-10p and PHYL-10q, both as part of larger chromosomes. The overall data indicate that, contrary to common opinion, PHYL-10p and PHYL-10q were distinct chromosomes in the primate ancestor. Analysis of the Saimiri sciureus (SSC) PHYL-10q marker order showed that it was isosequential with the Callithrix jacchus PHYL-10q, as well as with the PHYL-10q platyrrhine ancestral form. The SSC centromere, nevertheless, was located in a different chromosomal region, therefore suggesting that a centromeric repositioning event occurred in this species.     

Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.     

Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.     

Biological insights in the pathogenesis of hypermobile Ehlers-Danlos syndrome from proteome profiling of patients' dermal myofibroblasts

Hypermobile Ehlers-Danlos syndrome (hEDS), mainly characterized by generalized joint hypermobility and its complications, minor skin changes, and apparently segregating with an autosomal dominant pattern, is still without a known molecular basis. Hence, its diagnosis is only clinical based on a strict set of criteria defined in the revised EDS nosology. Moreover, the hEDS phenotypic spectrum is wide-ranging and comprises multiple associated signs and symptoms shared with other heritable or acquired connective tissue disorders and chronic inflammatory diseases. In this complex scenario, we previously demonstrated that hEDS patients' skin fibroblasts show phenotypic features of myofibroblasts, widespread extracellular matrix (ECM) disarray, perturbation of ECM-cell contacts, and dysregulated expression of genes involved in connective tissue architecture and related to inflammatory and pain responses. Herein, the cellular proteome of 6 hEDS dermal myofibroblasts was compared to that of 12 control fibroblasts to deepen the knowledge on mechanisms involved in the disease pathogenesis. Qualitative and quantitative differences were assessed based on top-down and bottom-up approaches and some differentially expressed proteins were proofed by biochemical analyses. Proteomics disclosed the differential expression of proteins principally implicated in cytoskeleton organization, energy metabolism and redox balance, proteostasis, and intracellular trafficking. Our findings offer a comprehensive view of dysregulated protein networks and related pathways likely associated with the hEDS pathophysiology. The present results can be regarded as a starting point for future in-depth investigations aimed to decipher the functional impact of potential bioactive molecules for the development of targeted management and therapies.     

Energy expenditure at rest and during walking in patients with chronic respiratory failure: a prospective two-phase case-control study

Background

Measurements of Energy Expenditure (EE) at rest (REE) and during physical activities are increasing in interest in chronic patients. In this study we aimed at evaluating the validity/reliability of the SenseWear®Armband (SWA) device in terms of REE and EE during assisted walking in Chronic Respiratory Failure (CRF) patients receiving long-term oxygen therapy (LTOT).

Methodology/Principal Findings

In a two-phase prospective protocol we studied 40 severe patients and 35 age-matched healthy controls. In phase-1 we determined the validity and repeatability of REE measured by SWA (REEa) in comparison with standard calorimetry (REEc). In phase-2 we then assessed EE and Metabolic Equivalents-METs by SWA during the 6-minute walking test while breathing oxygen in both assisted (Aid) or unassisted (No-Aid) modalities. When compared with REEc, REEa was slightly lower in patients (1351±169 vs 1413±194 kcal/day respectively, p<0.05), and less repeatable than in healthy controls (0.14 and 0.43 coefficient respectively). COPD patients with CRF patients reported a significant gain with Aid as compared with No-Aid modality in terms of meters walked, perceived symptoms and EE.

Conclusions/Significance

SWA provides a feasible and valid method to assess the energy expenditure in CRF patients on LTOT, and it shows that aided walking results in a substantial energy saving in this population.     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

3-hydroxykynurenine as a substrate/activator for mushroom tyrosinase

3-Hydroxykynurenine is a tryptophan metabolite with an o-aminophenol structure. It is both a tyrosinase activator and a substrate, reducing the lag phase, stimulating the monophenolase activity, and being oxidized to xanthommatin. In the early stage of monophenol hydroxylation, catechol accumulation takes place, whereas 3-hydroxykynurenine is substantially unchanged and no significant amounts of the o-quinone are produced. These results suggest an activating action of 3-hydroxykynurenine toward o-hydroxylation of monophenols. 3-Hydroxykynurenine could therefore well act as a physiological device to control phenolics metabolism to catechols and quinonoids.     

In Type 1 Diabetes a Subset of Anti-Coxsackievirus B4 Antibodies Recognize Autoantigens and Induce Apoptosis of Pancreatic Beta Cells

Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients’sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients’ sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.