116 research outputs found
Reconstitution of a Minimal DNA Replicase From Pseudomonas Aeruginosa and Stimulation by Non-Cognate Auxiliary Factors
DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (β), single-stranded DNA-binding protein, a complex containing the polymerase (α) and exonuclease (ϵ) subunits, and the essential components of the DnaX complex (τ3δδ′). Efficient primer elongation requires the presence of αϵ, β, and τ3δδ′. Pseudomonas aeruginosa αϵ can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas β and τ3δδ′ exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli ψ subunit was not apparent in sequence similarity searches, addition of purified E. coli χ and ψ (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas τ3δδ′ complex ∼10-fold and enables the reconstituted enzyme to function better under physiological salt conditions
Discovery and Characterization of the Cryptic Psi Subunit of the Pseudomonad DNA Replicase
We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of α and ϵ (polymerase and editing nuclease), β (processivity factor), and the essential τ, δ, and δ′ components of the clamp loader complex (Jarvis, T., Beaudry, A., Bullard, J., Janjic, N., and McHenry, C. (2005) J. Biol. Chem. 280, 7890-7900). In Escherichia coli DNA polymerase III holoenzyme, χ and Ψ are tightly associated clamp loader accessory subunits. The addition of E. coli χΨ to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of χ and Ψ counterparts in P. aeruginosa. The P. aeruginosa χ subunit was recognizable from sequence similarity, but Ψ was not. Here we report purification of an endogenous replication complex from P. aeruginosa. Identification of the components led to the discovery of the cryptic Ψ subunit, encoded by holD. P. aeruginosa χ and Ψ were co-expressed and purified as a 1:1 complex. P. aeruginosa χΨ increased the specific activity of τ3δδ′ 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between χΨ and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa Ψ allowed us to identify Ψ subunits from several other Pseudomonads and to predict probable translational start sites for this protein family. This represents the first identification of a highly divergent branch of the Ψ family and confirms the existence of Ψ in several organisms in which Ψ was not identifiable based on sequence similarity alone
Giant Cell Tumor: A Rare Condition in the Immature Skeleton—A Retrospective Study of Symptoms, Treatment, and Outcome in 16 Children
Evaluation of Pyridoacridine Alkaloids in a Zebrafish Phenotypic Assay
Three new minor components, the pyridoacridine alkaloids 1-hydroxy-deoxyamphimedine (1), 3-hydroxy-deoxyamphimedine (2), debromopetrosamine (3), and three known compounds, amphimedine (4), neoamphimedine (5) and deoxyamphimedine (6), have been isolated from the sponge Xestospongia cf. carbonaria, collected in Palau. Structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1–6 were evaluated in a zebrafish phenotype-based assay. Amphimedine (4) was the only compound that caused a phenotype in zebrafish embryos at 30 μM. No phenotype other than death was observed for compounds 1–3, 5, 6
A New Diketopiperazine, Cyclo-(4-S-hydroxy-R-proline-R-isoleucine), from an Australian Specimen of the Sponge Stelletta sp. â€
While investigating the cytotoxic activity of the methanol extract of an Australian marine sponge Stelletta sp. (Demospongiae), a new diketopiperazine, cyclo-(4-S-hydroxy-R-proline-R-isoleucine) (1), was isolated together with the known bengamides; A (2), F (3), N (4), Y (5), and bengazoles; Z (6), C4 (7) and C6 (8). The isolation and structure elucidation of the diketopiperazine (1), together with the activity of 1–8 against a panel of human and mammalian cell lines are discussed
Accelerating and Retarding Anomalous Diffusion
In this paper Gaussian models of retarded and accelerated anomalous diffusion
are considered. Stochastic differential equations of fractional order driven by
single or multiple fractional Gaussian noise terms are introduced to describe
retarding and accelerating subdiffusion and superdiffusion. Short and long time
asymptotic limits of the mean squared displacement of the stochastic processes
associated with the solutions of these equations are studied. Specific cases of
these equations are shown to provide possible descriptions of retarding or
accelerating anomalous diffusion.Comment: 18 page, 1 figur
Aptamer-based multiplexed proteomic technology for biomarker discovery
Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine
A fast and accurate energy source emulator for wireless sensor networks
The capability to either minimize energy consumption in battery-operated devices, or to adequately exploit energy harvesting from various ambient sources, is central to the development and engineering of energy-neutral wireless sensor networks. However, the design of effective networked embedded systems targeting unlimited lifetime poses several challenges at different architectural levels. In particular, the heterogeneity, the variability, and the unpredictability of many energy sources, combined to changes in energy required by powered devices, make it difficult to obtain reproducible testing conditions, thus prompting the need of novel solutions addressing these issues. This paper introduces a novel embedded hardware-software solution aimed at emulating a wide spectrum of energy sources usually exploited to power sensor networks motes. The proposed system consists of a modular architecture featuring small factor form, low power requirements, and limited cost. An extensive experimental characterization confirms the validity of the embedded emulator in terms of flexibility, accuracy, and latency while a case study about the emulation of a lithium battery shows that the hardware-software platform does not introduce any measurable reduction of the accuracy of the model. The presented solution represents therefore a convenient solution for testing large-scale testbeds under realistic energy supply scenarios for wireless sensor networks
Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response
Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-Dk and the MCMV m04 protein. Using H2 chimeras between H2-Db and -Dk, we demonstrate that the H2-Dk peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-Dk, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor
Six sequence variants on chromosome 9p21.3 are associated with a positive family history of myocardial infarction: a multicenter registry
<p>Abstract</p> <p>Background</p> <p>Recent genome-wide association studies have identified several genetic loci linked to coronary artery disease (CAD) and myocardial infarction (MI). The 9p21.3 locus was verified by numerous replication studies to be the first common locus for CAD and MI. In the present study, we investigated whether six single nucleotide polymorphisms (SNP) rs1333049, rs1333040, rs10757274, rs2383206, rs10757278, and rs2383207 representing the 9p21.3 locus were associated with the incidence of an acute MI in patients with the main focus on the familial aggregation of the disease.</p> <p>Methods</p> <p>The overall cohort consisted of 976 unrelated male patients presenting with an acute coronary syndrome (ACS) with ST-elevated (STEMI) as well as non-ST-elevated myocardial infarction (NSTEMI). Genotyping data of the investigated SNPs were generated and statistically analyzed in comparison to previously published findings of matchable control cohorts.</p> <p>Results</p> <p>Statistical evaluation confirmed a highly significant association of all analyzed SNP's with the occurrence of MI (p < 0.0001; OR: 1.621-2.039). When only MI patients with a positive family disposition were comprised in the analysis a much stronger association of the accordant risk alleles with incident disease was found with odds ratios up to 2.769.</p> <p>Conclusions</p> <p>The findings in the present study confirmed a strong association of the 9p21.3 locus with MI particularly in patients with a positive family history thereby, emphasizing the pathogenic relevance of this locus as a common genetic cardiovascular risk factor.</p
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