Structure of an atypical periplasmic adaptor from a multidrug efflux pump of the spirochete Borrelia burgdorferi

Periplasmic adaptor proteins are essential components of bacterial tripartite multidrug efflux pumps. Here we report the 2.35 Å resolution crystal structure of the BesA adaptor from the spirochete Borrelia burgdorferi solved using selenomethionine derivatized protein. BesA shows the archetypal linear, flexible, multi-domain architecture evident among proteobacteria and retains the lipoyl, β-barrel and membrane-proximal domains that interact with the periplasmic domains of the inner membrane transporter. However, it lacks the α-hairpin domain shown to establish extensive coiled-coil interactions with the periplasmic entrance helices of the outer membrane-anchored TolC exit duct. This has implications for the modelling of assembled tripartite efflux pumps.     

Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastatic cells. This notion is supported by preclinical findings that inhibition of pro-lymphangiogenic signaling during tumor development reduces cell spread to sentinel lymph nodes (SLNs). However, it is unclear how lymphatics downstream of SLNs contribute to metastatic spread into distal organs, or if modulating distal lymph transport impacts disease progression. Utilizing murine models of metastasis, longitudinal in vivo imaging of lymph transport, and function blocking antibodies against two VEGF family members, we provide evidence that distal lymphatics undergo disease course-dependent up-regulation of lymph transport coincidental with structural remodeling. Inhibition of VEGF-C activity with antibodies against VEGF-C or NRP2 prevented these disease-associated changes. Furthermore, utilizing a novel model of adjuvant treatment, we demonstrate that antagonism of VEGF-C or NRP2 decreases post SLN metastasis. These data support a potential therapeutic strategy for inhibiting distant metastatic dissemination via targeting tumor-associated lymphatic remodeling.     

Quantification of urinary zwitterionic organic acids using weak-anion exchange chromatography with tandem MS detection

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r2>or=0.995), accuracy (85-115%), precision (C.V.<12%), sample preparation stability (相似文献   

The influence of the exocyclic pyrimidine 5-methyl group on DNAse I cleavage and sequence recognition by drugs

Incorporation of modified nucleotides into DNA, using the PCR, has allowed us to probe the influence that the exocyclic 5-methyl group of pyrimidines has on DNAse I cleavage and sequence recognition by drugs. The results show that removal of the methyl group from the major groove, made possible by substituting uridine for thymidine, allows DNAse I to cleave more readily at AT-rich regions compared to normal DNA. By contrast, addition of an extra methyl group, contrived by substituting 5-methylcytidine for normal cytidine, allows DNAse I to cleave more readily at GC-rich regions compared to normal DNA. In the cutting pattern of DNA containing both uridine and 5-methyl cytosine, we find the cleavage characteristics of both the single-substituted DNA species combined. Thus, the presence or absence of the exocyclic 5-methyl group in the major groove has a strong influence on the relative intensity of cleavage of phosphodiester bonds by DNAse I. These nucleotide substitutions can also influence the sequence-selective binding of drugs to DNA. Whereas removal of the methyl group (replacement of T with U) generally has little effect on sequence recognition by a variety of drugs, addition of a methyl group (replacement of C with M) generates new binding sites for some intercalators, namely daunomycin, DACA and SN16713.     

Vegetation responses to landscape structure at multiple scales across a Northern Wisconsin,USA, pine barrens landscape

Increasing awareness of the importance of scale and landscape structure to landscape processes and concern about loss of biodiversity has resulted in efforts to understand patterns of biodiversity across multiple scales. We examined plant species distributions and their relationships to landscape structure at varying spatial scales across a pine barrens landscape in northern Wisconsin, U.S.A. We recorded plant species cover in 1×1 m plots every 5 m along a 3575 m transect, along with variables describing macro- and micro-landscape structure. A total of 139 understory plant species were recorded. The distributions of many species appeared to be strongly associated with landscape structural features, such as distinct management patches and roads. TWINSPAN and detrended correspondence analysis (DCA) identified three groups of species that overlapped extensively in the ordination, possibly reflecting the relatively homogeneous nature of disturbance in the pine barrens landscape. Distribution of understory plants did not reflect all of the patch types we identified along the transect; plot ordination and classification resulted in three to five plot groups that differed in niche breadth. Wavelet transforms showed varying relationships between landscape features and plant diversity indices (Shannon–Weiner, Simpson's Dominance) at different resolutions. Wavelet variances indicated that patterns of Shannon diversity were dominated by coarse resolutions ranging from 900–1500 m, which may have been related to topography. Patterns of Simpson's Dominance were dominated by 700 m resolution, possibly associated with canopy cover. However, a strong correspondence between overstory patch type and diversity was found for several patch types at ranges of scales that varied by patch type. Effects of linear features such as roads were apparent in the wavelet transforms at resolutions of about 5–1000 m, suggesting roads may have an important impact on plant diversity at landscape scales. At broad scales, landscape context appeared more important to diversity than individual patches, suggesting that changes in structure at fine resolutions could alter overall diversity characteristics of the landscape. Therefore, a hierarchical perspective is necessary to recognize potential large-scale change resulting from small-scale activities.     

Phylogenetic relationships in Nuphar (Nymphaeaceae): evidence from morphology,chloroplast DNA,and nuclear ribosomal DNA

The genus Nuphar consists of yellow-flowered waterlilies and is widely distributed in north-temperate bodies of water. Despite regular taxonomic evaluation of these plants, no explicit phylogenetic hypotheses have been proposed for the genus. We investigated phylogenetic relationships in Nuphar using morphology and sequences of the chloroplast gene matK and of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Two major lineages within Nuphar are consistently resolved with the morphological and molecular data sets. One lineage comprises New World taxa and the other represents a primarily Old World lineage. Relationships within the major lineages were poorly resolved by morphology and ITS, yet certain relationships were elucidated by all analyses. Most notable is the strong support for a monophyletic lineage of dwarf taxa and the alliance of the North American N. microphylla with the Eurasian taxa. Minor discordance between the independent cladograms is accounted for by hybridization. The common taxonomic practice of uniting all North American and Eurasian taxa under one species is not supported phylogenetically.     

The carbonate radical anion-induced covalent aggregation of human copper, zinc superoxide dismutase, and alpha-synuclein: intermediacy of tryptophan- and tyrosine-derived oxidation products

In this review, we describe the free radical mechanism of covalent aggregation of human copper, zinc superoxide dismutase (hSOD1). Bicarbonate anion (HCO3-) enhances the covalent aggregation of hSOD1 mediated by the SOD1 peroxidase-dependent formation of carbonate radical anion (CO3*-), a potent and selective oxidant. This species presumably diffuses out the active site of hSOD1 and reacts with tryptophan residue located on the surface of hSOD1. The oxidative degradation of tryptophan to kynurenine and N-formyl kynurenine results in the covalent crosslinking and aggregation of hSOD1. Implications of oxidant-mediated aggregation of hSOD1 in the increased cytotoxicity of motor neurons in amyotrophic lateral sclerosis are discussed.     

Molecular phylogeny of the hexagrammid fishes using a multi-locus approach

Ideally, organisms are grouped into monophyletic assemblages reflecting their evolutionary histories. Single (molecular) markers can reflect the evolutionary history of the marker, rather than the species in question, therefore, phylogenetic relationships should be inferred from adequate sampling of characters. Because the use of multiple loci greatly improves the resolving power of the molecular assay, we constructed a molecular phylogeny of the family Hexagrammidae based on six loci, including two mitochondrial and four nuclear loci. The resulting molecular phylogeny, from the combined data, was significantly different from the morphological topology suggested by Shinohara [Memoirs of the Faculty of Fisheries, Hokkaido University 41 (1994) 1]. Our data support a monophyletic assemblage for the genera Hexagrammos and Pleurogrammus. However, other taxa traditionally included in the family Hexagrammidae did not form a monophyletic assemblage. The monotypic genus Ophiodon was more closely associated with cottids than with other hexagrammids. Our data concur with the morphological topology in that the genera Zaniolepis and Oxylebius formed a monophyletic clade, which was distinct and basal to the remaining hexagrammids, seven cottids and one agonid.     

Iron porphyrin treatment extends survival in a transgenic animal model of amyotrophic lateral sclerosis

Oxidative damage, produced by mutant Cu/Zn superoxide dismutase (SOD1), may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating motor neuron degenerative disease. A novel approach to antioxidant therapy is the use of metalloporphyrins that catalytically scavenge a wide range of reactive oxygen and reactive nitrogen species. In this study, we examined the therapeutic potential of iron porphyrin (FeTCPP) in the G93A mutant SOD1 transgenic mouse model of ALS. We found that intraperitoneal injection of FeTCPP significantly improved motor function and extended survival in G93A mice. Similar results were seen with a second group of mice wherein treatment with FeTCPP was initiated at the onset of hindlimb weakness-roughly equivalent to the time at which treatment would begin in human patients. FeTCPP-treated mice also showed a significant reduction in levels of malondialdehyde (a marker of lipid peroxidation), in total content of protein carbonyls (a marker of protein oxidation), and increased neuronal survival in the spinal cord. These results therefore provide further evidence of oxidative damage in a mouse model of ALS, and suggest that FeTCPP could be beneficial for the treatment of ALS patients.