Alpha-synuclein pore forming activity upon membrane association

Alpha-synuclein is a natively unfolded protein widely expressed in neurons at the presynaptic level. It is linked to Parkinson's disease by two lines of evidence: amyloid fibrils of the protein accumulate in patients' brains and three genetic mutants cause autosomal dominant forms of the disease. The biological role of the protein and the mechanisms involved in the etiopathogenesis of Parkinson's disease are still unknown. Membrane binding causes the formation of an amphipathic alpha-helix, which lies on the surface without crossing the bilayer. Recent observations however reported that the application of a voltage induces a pore-like activity of alpha-synuclein. This study aims to characterize the pore forming activity of the protein starting from its monomeric form. In particular, experiments with planar lipid membranes allowed recording of conductance activity bursts with a defined and reproducible fingerprint. Additional experiments with deletion mutants and covalently bound alpha-synuclein dimers were performed to understand both pore assembly and stoichiometry. The information acquired allowed formulation of a model for pore formation at different conductance levels.     

Sphingomyelinase depresses force and calcium sensitivity of the contractile apparatus in mouse diaphragm muscle fibers

Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2',6'-dimethyl-Tyr-Lys-Phe-NH(2), decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m(2): control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus.     

Kinetics of muscle deoxygenation and microvascular PO(2) during contractions in rat: comparison of optical spectroscopy and phosphorescence-quenching techniques

The overarching presumption with near-infrared spectroscopy measurement of muscle deoxygenation is that the signal reflects predominantly the intramuscular microcirculatory compartment rather than intramyocyte myoglobin (Mb). To test this hypothesis, we compared the kinetics profile of muscle deoxygenation using visible light spectroscopy (suitable for the superficial fiber layers) with that for microvascular O(2) partial pressure (i.e., Pmv(O(2)), phosphorescence quenching) within the same muscle region (0.5～1 mm depth) during transitions from rest to electrically stimulated contractions in the gastrocnemius of male Wistar rats (n = 14). Both responses could be modeled by a time delay (TD), followed by a close-to-exponential change to the new steady level. However, the TD for the muscle deoxygenation profile was significantly longer compared with that for the phosphorescence-quenching Pmv(O(2)) [8.6 ± 1.4 and 2.7 ± 0.6 s (means ± SE) for the deoxygenation and Pmv(O(2)), respectively; P < 0.05]. The time constants (τ) of the responses were not different (8.8 ± 4.7 and 11.2 ± 1.8 s for the deoxygenation and Pmv(O(2)), respectively). These disparate (TD) responses suggest that the deoxygenation characteristics of Mb extend the TD, thereby increasing the duration (number of contractions) before the onset of muscle deoxygenation. However, this effect was insufficient to increase the mean response time. Somewhat differently, the muscle deoxygenation response measured using near-infrared spectroscopy in the deeper regions (～5 mm depth) (～50% type I Mb-rich, highly oxidative fibers) was slower (τ = 42.3 ± 6.6 s; P < 0.05) than the corresponding value for superficial muscle measured using visible light spectroscopy or Pmv(O(2)) and can be explained on the basis of known fiber-type differences in Pmv(O(2)) kinetics. These data suggest that, within the superficial and also deeper muscle regions, the τ of the deoxygenation signal may represent a useful index of local O(2) extraction kinetics during exercise transients.     

Impact of biocides on biofilm formation by methicillin-resistant Staphylococcus aureus (ST239-SCCmecIII) isolates

Procedures of sterilization and disinfection are essential to ensure that medical and surgical instruments will not transmit infectious pathogens to patients. In the present paper, we tested the residual effect of these compounds on biofilm formation and its efficiency in disrupting preformed biofilms using methicillin-resistant Staphylococcus aureus (MRSA) isolates of the lineage ST239-SCCmecIII. All compounds examined, except 70% alcohol, caused a significant impairment in biofilm formation with concomitant inhibition of cell growth. Among the compounds examined, 10% povidone-iodine (PVP-I) was the only antiseptic that exhibited more than 90% reduction of both biofilm formation and dispersion. In the group of sterilants and disinfectants, a formulation containing 7% hydrogen peroxide and 0.2% peracetic acid (HP-PA), and sodium hypochlorite with 1% active chlorine (NaOCl) were equally effective.     

A <Emphasis Type="Italic">de novo</Emphasis> germline mutation in <Emphasis Type="Italic">MYH7</Emphasis> causes a progressive dominant myopathy in pigs

Background

About 9% of the offspring of a clinically healthy Piétrain boar named ‘Campus’ showed a progressive postural tremor called Campus syndrome (CPS). Extensive backcross experiments suggested a dominant mode of inheritance, and the founder boar was believed to be a gonadal mosaic. A genome-scan mapped the disease-causing mutation to an 8 cM region of porcine chromosome 7 containing the MHY7 gene. Human distal myopathy type 1 (MPD1), a disease partially resembling CPS in pigs, has been associated with mutations in the MYH7 gene.

Results

The porcine MYH7 gene structure was predicted based on porcine reference genome sequence, porcine mRNA, and in comparison to the human ortholog. The gene structure was highly conserved with the exception of the first exon. Mutation analysis of a contiguous genomic interval of more than 22 kb spanning the complete MYH7 gene revealed an in-frame insertion within exon 30 of MYH7 (c.4320_4321insCCCGCC) which was perfectly associated with the disease phenotype and confirmed the dominant inheritance. The mutation is predicted to insert two amino acids (p.Ala1440_Ala1441insProAla) in a very highly conserved region of the myosin tail. The boar ‘Campus’ was shown to be a germline and somatic mosaic as assessed by the presence of the mutant allele in seven different organs.

Conclusion

This study illustrates the usefulness of recently established genomic resources in pigs. We have identified a spontaneous mutation in MYH7 as the causative mutation for CPS. This paper describes the first case of a disorder caused by a naturally occurring mutation in the MYH7 gene of a non-human mammalian species. Our study confirms the previous classification as a primary myopathy and provides a defined large animal model for human MPD1. We provide evidence that the CPS mutation occurred during the early development of the boar ‘Campus’. Therefore, this study provides an example of germline mosaicism with an asymptomatic founder.

     

Effects of a new photoactivatable cationic porphyrin on ciliated protozoa and branchiopod crustaceans, potential components of freshwater ecosystems polluted by pathogenic agents and their vectors

The increasing use of photosensitized processes for disinfection of microbiologically polluted waters requires a precise definition of the factors controlling the degree of photosensitivity in target and non-target organisms. In this regard, tests with protozoa and invertebrates which have a natural habitat in such waters may be used as first screening methods for the assessment of possible hazards for the ecosystem. A new cationic porphyrin, namely meso-tri(N-methyl-pyridyl)mono(N-dodecyl-pyridyl)porphine (C12), is tested in this work on the protozoan Ciliophora Colpoda inflata and Tetrahymena thermophila and the Crustacea Branchiopoda Artemia franciscana and Daphnia magna. The protocol involved 1 h incubation with porphyrin doses in the 0.1-10.0 μM range and subsequent irradiation with visible light at a fluence rate of 10 mW cm(-2). The results indicate that C12 porphyrin has a significant affinity for C. inflata and T. thermophila; this is also shown by fluorescence microscopic analyses. C. inflata cysts were resistant to the phototreatment up to a porphyrin dose of 0.6 μM. The effects of C12 on cysts have been evaluated at 3 and 24 h after the end of the phototreatment; a delay in the excystment process was observed. T. thermophila was fairly resistant to the phototreatment with C12 porphyrin. The data obtained with the two crustaceans indicated that the effects of dark- and photo-treatment with C12 need to be closely examined for every organism. A. franciscana is more resistant, probably owing to its ability to adapt to extreme conditions, while the high level of photosensitivity displayed by Daphnia magna represents a potential drawback, as this organism is often selected as a reference standard for assessing the environmental safety. Thus, while C12 photosensitisation can represent a useful tool for inducing a microbicidal or larvicidal action on polluted waters, the irradiation protocols must be carefully tailored to the nature of the specific water basin, and in particular to its biotic characteristics.     

Molecular phylogenetics of the Brazilian giant bromeliads (Alcantarea, Bromeliaceae): implications for morphological evolution and biogeography

The genus Alcantarea comprises near 30 species endemic to rocky outcrops from eastern Brazil. Most species are ornamental and several are threatened due to habitat loss and over collection. In this paper we examine the phylogenetics of Alcantarea and its relationship with the Brazilian members of Vriesea, a genus of which Alcantarea has been treated as a subgenus. We discuss the morphological evolution of the stamen position and its implication for pollination and the occurrence of Alcantarea in the Espinha?o mountain range rocky savanna-like habitat vegetation. DNA sequence data derived from two plastid markers (trnK-rps16, trnC-petN) and from a low copy nuclear gene (Floricaula/Leafy) together with 20 nuclear microsatellite loci were the data source to perform analyses and construct phylogenetic and Neighbor Joining trees for the genus. Alcantarea is well supported as monophyletic in both Bayesian and parsimony analyses, but sections of Vriesea, represented by the eastern Brazilian species, appear paraphyletic. Microsatellites delimit geographically isolated species groups. Nevertheless individuals belonging to a single species may appear related to distinct clusters of species, suggesting that hybridization and/or homoplasy and/or incomplete lineage sorting are also influencing the analysis based on such markers and may be the reasons for some unexpected results. Alcantarea brasiliana is hypothesized as putative hybrid between A. imperialis and A. geniculata. Spreading stamens, a morphological floral characteristic assumed to be related to Chiropterophily, apparently evolved multiple times within the genus, and invasion of rocky savanna-like habitat vegetation by Atlantic rainforest ancestors seems to have occurred multiple times as well.     

Differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma

Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.     

Durum wheat by-products as natural sources of valuable nutrients

This review reports the use of wheat milling by-products for the extraction of high quality oil and vitamin E including our results on the exploitation of durum wheat bran as a valuable source of important healthful compounds. Wheat oil can be used as an ingredient in food, pharmaceutical or cosmetic preparations because it contains important bioactive compounds such as vitamin E, carotenoids and unsaturated fatty acids. Different methods are used for oil recovery from plant materials, such as solvent extraction, mechanical pressing or the eco-friendly supercritical carbon dioxide (SC-CO₂) extraction technology. By using SC-CO₂, we obtained an oil from durum wheat (Triticum durum Desf.) bran and optimized the extraction conditions to increase oil and vitamin E yields. Wheat bran, which is composed of pericarp, aleurone layer and germ, is discarded during the early stages of durum wheat milling processes to obtain a final product (semolina) that is stable over time. Maximum oil and vitamin E yields were obtained when a durum wheat bran matrix with particle size of ~30 mesh and a moisture content of 2.6 % was used. The optimal conditions for oil extraction were: 300–350 bar, 60–70 °C, and 4 l min^?1 gaseous CO₂ flow rate for 1 h. The chemical composition (vitamin E forms, carotenoids, quinones, lipids and fatty acids) of the SC-CO₂ extracted oil was analyzed and compared to that of the oil extracted by Soxhlet using hexane as solvent. The findings here reported highlight the importance of durum wheat bran as a rich source of valuable natural nutrients.