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Independent Confirmation (independent + confirmation)
Selected AbstractsOn-line optical and X-ray spectroscopies with crystallography: an integrated approach for determining metalloprotein structures in functionally well defined statesJOURNAL OF SYNCHROTRON RADIATION, Issue 5 2008Mark J. Ellis X-ray-induced redox changes can lead to incorrect assignments of the functional states of metals in metalloprotein crystals. The need for on-line monitoring of the status of metal ions (and other chromophores) during protein crystallography experiments is of growing importance with the use of intense synchrotron X-ray beams. Significant efforts are therefore being made worldwide to combine different spectroscopies in parallel with X-ray crystallographic data collection. Here the implementation and utilization of optical and X-ray absorption spectroscopies on the modern macromolecular crystallography (MX) beamline 10, at the SRS, Daresbury Laboratory, is described. This beamline is equipped with a dedicated monolithic energy-dispersive X-ray fluorescence detector, allowing X-ray absorption spectroscopy (XAS) measurements to be made in situ on the same crystal used to record the diffraction data. In addition, an optical microspectrophotometer has been incorporated on the beamline, thus facilitating combined MX, XAS and optical spectroscopic measurements. By uniting these techniques it is also possible to monitor the status of optically active and optically silent metal centres present in a crystal at the same time. This unique capability has been applied to observe the results of crystallographic data collection on crystals of nitrite reductase from Alcaligenes xylosoxidans, which contains both type-1 and type-2 Cu centres. It is found that the type-1 Cu centre photoreduces quickly, resulting in the loss of the 595,nm peak in the optical spectrum, while the type-2 Cu centre remains in the oxidized state over a much longer time period, for which independent confirmation is provided by XAS data as this centre has an optical spectrum which is barely detectable using microspectrophotometry. This example clearly demonstrates the importance of using two on-line methods, spectroscopy and XAS, for identifying well defined redox states of metalloproteins during crystallographic data collection. [source] Redox enzymes in the plant plasma membrane and their possible rolesPLANT CELL & ENVIRONMENT, Issue 12 2000A. Bérczi ABSTRACT Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low-molecular-mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K1), all of which are likely to participate in redox processes. A few enzymes have already been identified: Monodehydroascorbate reductase (EC 1.6.5.4) is firmly bound to the cytosolic surface of the PM where it might be involved in keeping both cytosolic and, together with a b -type cytochrome, apoplastic ascorbate reduced. A malate dehydrogenase (EC 1.1.1.37) is localized on the inner side of the PM. Several NAD(P)H-quinone oxidoreductases have been purified from the cytocolic surface of the PM, but their function is still unknown. Different forms of nitrate reductase (EC 1.6.6.1,3) are found attached to, as well as anchored in, the PM where they may act as a nitrate sensor and/or contribute to blue-light perception, although both functions are speculative. Ferric-chelate-reducing enzymes (EC 1.6.99.13) are localized and partially characterized on the inner surface of the PM but they may participate only in the reduction of ferric-chelates in the cytosol. Very recently a ferric-chelate-reducing enzyme containing binding sites for FAD, NADPH and hemes has been identified and suggested to be a trans -PM protein. This enzyme is involved in the reduction of apoplastic iron prior to uptake of Fe2+ and is induced by iron deficiency. The presence of an NADPH oxidase, similar to the so-called respiratory burst oxidase in mammals, is still an open question. An auxin-stimulated and cyanide-insensitive NADH oxidase (possibly a protein disulphide reductase) has been characterized but its identity is still awaiting independent confirmation. Finally, the only trans -PM redox protein which has been partially purified from plant PM so far is a high-potential and ascorbate-reducible b -type cytochrome. In co-operation with vitamin K1 and an NAD(P)H-quinone oxidoreductase, it may participate in trans -PM electron transport. [source] Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virusAUSTRALIAN VETERINARY JOURNAL, Issue 7 2004DB BOYLE Objective To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. Design Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. Results Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. Conclusion Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia. [source] The problem with "the Paleoptera Problem:" sense and sensitivityCLADISTICS, Issue 5 2003T.Heath Ogden While the monophyly of winged insects (Pterygota) is well supported, phylogenetic relationships among the most basal extant pterygote lineages are problematic. Ephemeroptera (mayflies) and Odonata (dragonflies) represent the two most basal extant lineages of winged insects, and determining their relationship with regard to Neoptera (remaining winged insects) is a critical step toward understanding insect diversification. A recent molecular analysis concluded that Paleoptera (Odonata Ephemeroptera) is monophyletic. However, we demonstrate that this result is supported only under a narrow range of alignment parameters. We have further tested the monophyly of Paleoptera using additional sequence data from 18SrDNA, 28S rDNA, and Histone 3 for a broader selection of taxa and a wider range of analytical methodologies. Our results suggest that the current suite of molecular data ambiguously resolve the three basal winged insect lineages and do not provide independent confirmation of Odonata + Neoptera as supported via morphological data. [source] GABRA2 and Alcohol Use Disorders: No Evidence of an Association in an Italian Case,Control StudyALCOHOLISM, Issue 4 2010Nicoletta Onori Background:, Alcoholism is a major health and social issue, a highly frequent disease and a cause of premature death. It is also the most expensive addictive disorder being related to high morbidity and mortality, violence, accidents, and social and legal problems. It is a quantitative disorder, where the combined incidence of environmental and multiple genetic factors varies from 1 subject to another. Recent association studies have identified several genes as candidates for alcoholism, including GABAA receptor genes, due to their role in mediating several behavioral effects of alcohol, such as motor incoordination, anxiolysis, sedation, and withdrawal. The proposed association between the 3, half of the gene encoding the alpha-2 subunit of GABA receptor (3,-GABRA2) and alcohol use disorders (AUDs) has received several independent confirmations. Methods:, In this study, 10 single nucleotide polymorphisms (SNPs) of the 3,-GABRA2 gene, previously reported to be implicated in alcohol dependence, were used to evaluate the linkage between selected SNPs and AUDs in an Italian sample and to compare findings with those of previous studies. Results:, No evidence of an association was found at the allele, genotype, haplotype, or diplotype levels between the 3,-GABRA2 polymorphisms investigated and alcoholism in 149 Italian alcoholics (98 alcohol dependents and 51 alcohol abusers) and 278 controls. Conclusions:, Despite previous reports, we did not find an association between AUDs and 3,-GABRA2 polymorphisms. This is probably due to the minimal comorbidity of our Italian sample suggesting that this gene is implicated in polysubstance dependence rather than in alcoholism alone. [source] | |||||||||||||