Human-mediated introgression of exotic chukar (Alectoris chukar, Galliformes) genes from East Asia into native Mediterranean partridges

Mediterranean red-legged (Alectoris rufa) and rock (Alectoris graeca) partridge populations are affected by genetic pollution. The chukar partridge (Alectoris chukar), a species only partly native to Europe, is the most frequently introgressive taxon detected in the genome of hybrid partridges. Both theoretical (evolutionary) and practical (resources management) matters spur to get insight into the geographic origin of the A. chukar hybridizing swarm. The phenotypic A. rufa populations colonizing the easternmost part of the distribution range of this species, the islands of Elba (Italy) and Corsica (France), were investigated. The analysis of both mitochondrial (mtDNA: Cytochrome-b gene plus Control Region: 2,250 characters) and nuclear (Short Tandem Repeats, STR; Random Amplified Polymorphic DNA, RAPD) genomes of 25 wild (Elba) and 20 captive (Corsica) partridges, disclosed spread introgression of chukar origin also in these populations. All mtDNA haplotypes of Elba and Corsica partridges along with those we obtained from other A. rufa (total, n = 111: Italy, Spain, France) and A. graeca (n = 6, Italy), were compared with the mtDNA haplotypes of chukars (n = 205) sampled in 20 countries. It was found that the A. chukar genes detected in red-legged (n = 43) and rock partridges (n = 4) of Spain, France and Italy as well as in either introduced (Italy) or native (Greece, Turkey) chukars (n = 35) were all from East Asia. Hence, a well-defined geographic origin of the exotic chukar genes polluting the genome of native Mediterranean A. rufa and A. graeca (inter-specific level) as well as A. chukar (intra-specific level), was demonstrated.     

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Brazil

Until 2000, efforts into organising tissue banks in Brazil had not progressed far beyond small “in house” tissue storage repositories, usually annexed to Orthopaedic Surgery Services. Despite the professional entrepreneurship of those working as part time tissue bankers in such operations, best practices in tissue banking were not always followed due to the lack of regulatory standards, specialised training, adequate facilities and dedicated personnel. The Skin Bank of the Plastic Surgery Department of the Hospital das Clinicas of Sao Paulo, the single skin bank in Brazil, was not an exception. Since 1956, restricted and unpredictable amounts of skin allografts were stored under refrigeration for short periods under very limited quality controls. As in most “tissue banks” at that time in Brazil, medical and nursing staff worked on a volunteer and informal basis undergoing no specific training. IAEA supported the implementation of the tissue banking program in Brazil through the regional project RLA/7/009 “Quality system for the production of irradiated sterilised grafts” (1998–2000) and through two interregional projects INT/6/049 “Interregional Centre of Excellence in Tissue Banking”, during the period 2002–2004 and INT/6/052 “Improving the Quality of Production and Uses of Radiation Sterilised Tissue Grafts”, during the period 2002–2004. In 2001–2002, the first two years of operation of the HC-Tissue Bank, 53 skin transplants were carried out instead of the previous 4–5 a year. During this period, 75 individuals donated skin tissue, generating approximately 90,000 cm² of skin graft. The IAEA program were of great benefit to Brazilian tissue banking which has evolved from scattered make shift small operations to a well-established, high quality tissue banking scenario.     

The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70^S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I₁^PP2A enhanced E4orf4-induced cell killing and G₂/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.Our research group and others have shown that the 114-residue product of early region E4 of human adenoviruses, termed E4orf4, induces p53-independent cell death in human tumor cells (24, 25, 34-36, 55) and in Saccharomyces cerevisiae (23, 53). E4orf4 protein, which shares no obvious homology with other viral or cellular products, kills a wide range of human cancer cells but is believed to have reduced activity against normal human primary cells (6, 55, 56). Although in some cases E4orf4-expressing cells exhibit characteristics typical of apoptosis, including the presence of irregularly shaped and shrunken nuclei, cytoplasmic vacuolization, and membrane blebbing (24, 25, 50, 55), cell death may more typically be independent of caspase activation (24, 25, 30, 32, 50). With H1299 human non-small-cell lung carcinoma cells, death is characterized by rapid cell rounding, enlargement, release from the surface of culture plates, cell cycle arrest in G₂/M and possibly G₁, and eventually, after an extended period, loss of membrane integrity (30). Both cytoplasmic and nuclear pathways have been observed, the former involving interactions with c-Src family kinases, activation of calpain, and remodeling of the actin cytoskeleton (7, 24, 50, 51, 58). Little is known about the nuclear pathway, which may represent the predominant death-inducing process. Our current evidence suggests that H1299 cells die following prolonged irreversible cell cycle arrest leading to mitotic catastrophe and death by a necrosis-like process (30).E4orf4 is known to associate with the Bα regulatory subunit of protein phosphatase 2A (PP2A) (22, 34), and this interaction appears to be necessary for the majority of E4orf4 toxicity in both yeast (23, 53) and human tumor cells (34, 56). PP2A is an abundant serine-threonine phosphatase involved in regulation of metabolism, splicing, translation, morphogenesis, development, and cell cycle progression (15, 19, 27, 43, 59). PP2A holoenzymes exist as multiple heterotrimeric complexes composed of a catalytic C subunit, an A subunit that functions as a scaffold, and a B-type regulatory subunit. Two forms each of the A and C subunits exist in mammalian cells; however, more than 20 B-type subunits have been identified in three unique classes (B/B55, B′/B56, B″/PR72), plus striatin/SG2NA (sometimes called B‴) (10, 19, 26). Although one group has suggested that E4orf4 protein interacts with one or more members of the B′/B56 class (57), it is generally accepted that interaction with the Bα/B55 subunit (Cdc55 in yeast) is important for induction of cell death in both human tumor cells and yeast (53, 57). Interestingly, a recent report has also suggested that in yeast, growth suppression induced by E4orf4 is mediated only in part by the catalytic C subunit of PP2A (31).In the present report, we show that E4orf4 protein interacts uniquely with members of the B55 class of PP2A B-type subunits, and at sufficient concentrations, it appears to become toxic by reducing dephosphorylation of substrates of B55-containing PP2A holoenzymes. As cell death is preceded by cell cycle arrest, we believe that key substrates may include proteins required for cell cycle progression.     

Solution Structure of Human Cardiac Troponin C in Complex with the Green Tea Polyphenol, (?)-Epigallocatechin 3-Gallate

Heart muscle contraction is regulated by Ca²⁺ binding to the thin filament protein troponin C. In cardiovascular disease, the myofilament response to Ca²⁺ is often altered. Compounds that rectify this perturbation are of considerable interest as therapeutics. Plant flavonoids have been found to provide protection against a variety of human illnesses such as cancer, infection, and heart disease. (−)-Epigallocatechin gallate (EGCg), the prevalent flavonoid in green tea, modulates force generation in isolated guinea pig hearts (Hotta, Y., Huang, L., Muto, T., Yajima, M., Miyazeki, K., Ishikawa, N., Fukuzawa, Y., Wakida, Y., Tushima, H., Ando, H., and Nonogaki, T. (2006) Eur. J. Pharmacol. 552, 123–130) and in skinned cardiac muscle fibers (Liou, Y. M., Kuo, S. C., and Hsieh, S. R. (2008) Pflugers Arch. 456, 787–800; and Tadano, N., Yumoto, F., Tanokura, M., Ohtsuki, I., and Morimoto, S. (2005) Biophys. J. 88, 314a). In this study we describe the solution structure of the Ca²⁺-saturated C-terminal domain of troponin C in complex with EGCg. Moreover, we show that EGCg forms a ternary complex with the C-terminal domain of troponin C and the anchoring region of troponin I. The structural evidence indicates that the binding site of EGCg on the C-terminal domain of troponin C is in the hydrophobic pocket in the absence of troponin I, akin to EMD 57033. Based on chemical shift mapping, the binding of EGCg to the C-terminal domain of troponin C in the presence of troponin I may be to a new site formed by the troponin C·troponin I complex. This interaction of EGCg with the C-terminal domain of troponin C·troponin I complex has not been shown with other cardiotonic molecules and illustrates the potential mechanism by which EGCg modulates heart contraction.Cardiovascular disease (CVD)² is the number one cause of morbidity and mortality in western culture. In the United States, ∼1 in 3 deaths in 2004 were caused by CVD (1). In heart failure, the ability of the heart to distribute blood throughout the body is perturbed, and there is a growing interest to develop drugs that directly regulate the response of the myofilament to Ca²⁺. Regulation of muscle contraction is triggered by Ca²⁺ binding to troponin. The troponin complex is situated at regular intervals along the thin filament, which is made up of two elongated polymers, f-actin and tropomyosin. The backbone of the thin filament is composed of actin molecules arranged in a double helix with tropomyosin wound around actin as a coiled-coil. Anchored at every seventh actin molecule is the heterotrimeric troponin complex, which consists of troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnC is the Ca²⁺-binding subunit of troponin and has four EF-hand helix-loop-helix motifs. TnI is the inhibitory subunit of troponin. It regulates the actin-myosin cross-bridge formation by flipping between TnC and actin in a Ca²⁺-dependent manner. At low levels of cytosolic Ca²⁺, TnI is bound to actin, causing tropomyosin to sterically block the binding of the actomyosin cross-bridges. On the other hand, when Ca²⁺ concentration is high, TnI translocates from actin to TnC inducing tropomyosin to change its orientation on actin so that the actin-myosin interaction may occur. The subunit TnT fetters the troponin complex to the thin filament by way of its association with TnI (for reviews on contraction see Refs. 2–5).The large number of structural studies on troponin and the thin filament has helped gain insight into the molecular mechanism of muscle contraction. TnC is a dumbbell-shaped protein that consists of terminal domains connected by an elongated flexible linker, as shown by solution NMR (6). The overall folds of the terminal domains of skeletal TnC (sTnC) and cardiac TnC (cTnC) are very similar (7–9). The apo state of the N-domain of sTnC (sNTnC) and cTnC (cNTnC) reveals that the domain is in a “closed” conformation, such that the hydrophobic core of the protein is buried (8, 10, 11). In the skeletal system, sNTnC “opens” when two Ca²⁺ ions bind (8, 10, 11). Alternatively, cNTnC contains only one functional Ca²⁺-binding site, and its global conformation does not change as significantly as in sNTnC (11). Nonetheless, Ca²⁺ binding promotes the association of the switch region of cTnI (residues 147–163) with cNTnC. cTnI-(147–163) forms an α-helix when associated with cNTnC and has been elucidated by NMR in the solution structure of cNTnC·Ca²⁺·cTnI-(147–163) (12) and by the x-ray crystallography structure of cTnC·3Ca²⁺·cTnI·-(31–210)·cTnT-(183–288) (13). The interaction of cTnI-(147–163) with cNTnC·Ca²⁺ is essential to draw the inhibitory (cTnI-(128–147)) and C-terminal (cTnI-(163–210)) regions of cTnI away from actin. cTnI-(128–147) is not visualized in the cardiac structure, probably due to disorder (13). In the skeletal crystal structure of sTnC·4Ca²⁺·sTnI-(1–182)·sTnT-(156–262), however, the inhibitory region of sTnI is visualized and makes electrostatic contacts with the central helix connecting the N- and C-terminal lobes of cTnC (14). The C-domain (CTnC) of both sTnC and cTnC has two functional binding sites for Ca²⁺ and remains largely unstructured without Ca²⁺ bound. The folding of this domain occurs in the presence of Ca²⁺ (15, 16). Throughout the relaxation-contraction cycle, cCTnC is Ca²⁺-saturated with both Ca²⁺-binding sites occupied (cCTnC·2Ca²⁺) and is associated with the anchoring region of cTnI (cTnI-(34–71)). The crystal structure of cTnC·3Ca²⁺·cTnI·-(31–210)·cTnT-(183–288) shows cTnI-(34–71) is α-helical when bound with cCTnC·2Ca²⁺(13). The interaction of cCTnC·2Ca²⁺ with cTnI-(34–71) is the primary site in which cTnC is tethered to the thin filament.In light of the importance of the Ca²⁺-dependent cTnI-cTnC interaction in the signaling of muscle contraction, the design of drugs that modulate this interaction would be useful in the treatment of heart disease. Compounds that treat CVD through modulation of the activity of cTnC are called Ca²⁺ sensitizers or desensitizers, depending on whether they positively or negatively influence its function. These drugs are safer than other currently prescribed medicines that alter the cytosolic Ca²⁺ homeostasis (such as milrinone and dobutamine), which may cause arrhythmia or death with prolonged usage.The potential therapeutic advantage of Ca²⁺ (de)sensitizers has led to the development of a number of compounds that target cTnC. Compounds have been identified that elicit their activity through binding either cNTnC or cCTnC. Levosimendan and pimobendan are examples of molecules that increase heart muscle contractility through binding to cNTnC. Conversely, the molecule W7 decreases contractility via its interaction with cNTnC. For recent reviews on the molecular mechanism of these compounds and others see Refs. 17–19. The discovery of small molecules that bind to cCTnC to elicit their Ca²⁺-sensitizing effects suggests that cCTnC is also a suitable target for the development of therapeutics. The Ca²⁺ sensitizer, EMD 57033, is approved for the treatment for heart failure in dogs and binds to cCTnC·2Ca²⁺(20). In the NMR structure of cCTnC·2Ca²⁺·EMD 57033, EMD 57033 is associated in the hydrophobic cavity of cCTnC·2Ca²⁺ (21). The interaction of EMD 57033 with cCTnC is stereospecific for the (+)-enantiomer and explains why the (−)-enantiomer is inactive (22). Because EMD 57033 has been shown to bind cCTnC·2Ca²⁺ concurrently with cTnI-(128–147) but not with cTnI-(34–71) (23), one postulate is that EMD 57033 acts as a Ca²⁺ sensitizer by weakening the interaction of cTnI-(34–71) with cCTnC·2Ca²⁺, thus increasing the propensity of cTnI-(128–147) to bind cCTnC·2Ca²⁺ in vivo. The dilated cardiomyopathy (DCM) mutation, G159D, of cCTnC has renewed interest in the role of the C-lobe for regulation in contraction. The mutation has been identified to decrease the sensitivity of the thin filament to Ca²⁺ (24). The source of the DCM phenotype of G159D might come from the modulation of the interaction of cCTnC·2Ca²⁺ with cTnI-(34–71) (25).Green tea (Camellia sinensis) is one of the most widely consumed beverages in the world, and several epidemiological studies have linked the consumption of tea with a decrease in CVD (26, 27). (−)-Epigallocatechin gallate (EGCg) is a polyphenol that exists abundantly in unfermented teas and has been identified as a modulator of heart contraction through its interaction with cTnC (28–30). Here we use NMR spectroscopy to elucidate the three-dimensional structure of the cCTnC·2Ca²⁺·EGCg complex. The solution structure reveals that EGCg binds at the hydrophobic core of cCTnC inducing a small structural “opening.” We also use two-dimensional NMR spectroscopy to monitor the binding of EGCg to cCTnC·2Ca²⁺ and cCTnC·2Ca²⁺·cTnI-(34–71). Because EGCg and cTnI-(34–71) can bind cCTnC concurrently, the inotropic effect of EGCg may stem from its modulation of the cTnI-(34–71)-cCTnC·2Ca²⁺ interaction. The solution structure of cCTnC·2Ca²⁺·EGCg provides insight into the mechanism in which EGCg might influence heart contraction. These results taken with previous research on the Ca²⁺ sensitizer EMD 57033 and the DCM mutation G159D bring into question the dogma that cNTnC is the exclusive site for regulation of contraction in cTnC.     

Chemical Composition and Antifungal Activity of the Essential Oils of Lavandula pedunculata (Miller) Cav.

The chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller ) Cav. , harvested in North and Central Portugal, were investigated. The essential oils were isolated by hydrodistillation and analyzed by GC and GC/MS. The minimal‐inhibitory concentration (MIC) and the minimal‐lethal concentration (MLC) of the essential oils and of their major constituents were used to evaluate the antifungal activity against different strains of fungi involved in candidosis, dematophytosis, and aspergillosis. The oils were characterized by a high percentage of oxygenated monoterpenes, the main compounds being 1,8‐cineole (2.4–55.5%), fenchone (1.3–59.7%), and camphor (3.6–48.0%). Statistical analysis differentiated the essential oils into two main types, one characterized by the predominance of fenchone and the other one by the predominance of 1,8‐cineole. Within the 1,8‐cineole chemotype, two subgroups were well‐defined taking into account the percentages of camphor. A significant antifungal activity of the oils was found against dermatophyte strains. The essential oil with the highest content of camphor was the most active with MIC and MLC values ranging from 0.32–0.64 μl/ml.     

The development of 3-D, in vitro, endothelial culture models for the study of coronary artery disease

The response of the vascular endothelium to wall shear stress plays a central role in the development and progression of atherosclerosis. Current studies have investigated endothelial response using idealized in vitro flow chambers. Such cell culture models are unable to accurately replicate the complex in vivo wall shear stress patterns arising from anatomical geometries. To better understand this implication, we have created both simplified/tubular and anatomically realistic in vitro endothelial flow models of the human right coronary artery. A post-mortem vascular cast of the human left ventricular outflow tract was used to create geometrically accurate silicone elastomer models. Straight, tubular models were created using a custom made mold. Following the culture of human abdominal aortic endothelial cells within the inner lumen, cells were exposed to steady flow (Re = 233) for varying time periods. The resulting cell morphology was analyzed in terms of shape index and angle of orientation relative to the flow direction. In both models a progressive elongation and alignment of the endothelium in the flow direction was observed following 8, 12, and 24 hours. This change, however, was significantly less pronounced in the anatomical model (as observed from morphological variations indicative of localized flow features). Differences were also observed between the inner and outer walls at the disease-prone proximal region. Since morphological adaptation is a visual indication of endothelial shear stress activation, the use of anatomical models in endothelial genetic and biochemical studies may offer better insight into the disease process.     

A prospective study of androgen levels, hormone-related genes and risk of rheumatoid arthritis

Introduction

Since current treatment options for patients suffering from active rheumatoid arthritis (RA) remain inadequate, especially for those unresponsive to disease-modifying antirheumatic drugs (DMARDs), new and improved medication is needed. This study evaluates the safety and efficacy of masitinib (AB1010), a potent and selective protein tyrosine kinase inhibitor of c-KIT, in the monotherapy treatment of DMARD-refractory RA.

Methods

This was a multicentre, uncontrolled, open-label, randomised, dose-ranging, phase 2a trial. Masitinib was administered orally to 43 patients who had inadequate response to DMARDs, at initial randomised dosing levels of 3 and 6 mg/kg per day over a 12-week period. Dose adjustment was permitted based upon tolerability and response criteria. Efficacy was assessed via American College of Rheumatology 20%/50%/70% improvement criteria (ACR20/50/70) responses, disease activity score using 28 joint counts (DAS28), index of improvement in RA (ACRn) and C-reactive protein (CRP) improvement, relative to baseline at week 12.

Results

Improvement was observed in all efficacy endpoints, including ACR20/50/70 scores of 54%, 26% and 8%, respectively, and a reduction in CRP level by greater than 50% for approximately half the population. This improvement was sustainable throughout an extension phase (> 84 weeks) and was also independent of initial DMARD resistance (anti-tumour necrosis factor-alpha and/or methotrexate). A relatively high patient withdrawal rate (37%) required the use of last observation carried forward (LOCF) data imputation. Incidence of adverse events was high (95%), although the majority were of mild or moderate severity with a considerable decline in frequency observed after 12 weeks of treatment. Two nonfatal serious adverse events were reported. Dose-response analyses tentatively indicate that an initial dosing level of 6.0 mg/kg per day administered orally in two daily intakes is the most appropriate, based upon potency and tolerability trends.

Conclusions

Treatment with masitinib improved DMARD-refractory active RA. Following an initial high incidence of mostly mild to moderate side effects during the first 12 weeks of treatment, masitinib appears to be generally well tolerated. This, together with evidence of a sustainable efficacy response, suggests that masitinib is suitable for long-term treatment regimens. Since this was the first study of masitinib in a nononcologic pathology, the relatively high patient withdrawal rate observed can be partly attributed to a highly cautious response to adverse events. There is sufficient compelling evidence to warrant further placebo-controlled investigation.

Trial registration

ClinicalTrials.gov NCT00831922.     

Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing,Including a Splice-Site Mutation in Nucleoredoxin

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.