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71.
Bacterial artificial chromosome (BAC) libraries are an important tool for positional cloning, gene analysis and physical mapping. During studies using BAC clones, it is often necessary to organize them into contiguous sequences (contigs). To finalize, join and extend the contigs, both cloning and sequencing of the ends of the inserts are required. Here, we describe a low-cost, accessible, fast and powerful method for the routine isolation of BAC ends. This method allows the isolation of 20 BAC clone ends in one day. The analysis of the ends reveals fragment sizes compatible with sequencing, and the structure of these clones allows the sequencing of both ends using the same plasmid. Moreover, long end fragments can be sequenced in both directions. 相似文献
72.
We present an analysis of birth seasonality in nine geographical regions within Austria for two time periods, 1881-1912 and 1947-1959. In the early period, geography, climate, and agricultural patterns were related to birth seasonality. By the latter time period, these factors were no longer related to birth seasonality. We propose a "resilience hypothesis," which suggests two levels of causal influences on birth seasonality. First, underlying the three significant features of birth seasonality patterns around the world are only a small number of major causes. But, second, there are a multiplicity of minor causes that result in small perturbations in these otherwise resilient and consistent patterns. 相似文献
73.
Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice 下载免费PDF全文
Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptor-destroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication. 相似文献
74.
Reovirus-induced apoptosis requires activation of transcription factor NF-kappaB 总被引:5,自引:0,他引:5 下载免费PDF全文
Connolly JL Rodgers SE Clarke P Ballard DW Kerr LD Tyler KL Dermody TS 《Journal of virology》2000,74(7):2981-2989
75.
Karim M ElSawy Adelene Sim David P Lane Chandra S Verma Leo SD Caves 《Cell cycle (Georgetown, Tex.)》2015,14(2):179-188
The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development. 相似文献
76.
Hannah Priyadarshini Gideon JiaYao Phuah Amy J. Myers Bryan D. Bryson Mark A. Rodgers M. Teresa Coleman Pauline Maiello Tara Rutledge Simeone Marino Sarah M. Fortune Denise E. Kirschner Philana Ling Lin JoAnne L. Flynn 《PLoS pathogens》2015,11(1)
Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas. 相似文献
77.
Jonathan A. Coles Elmarie Myburgh Ryan Ritchie Alana Hamilton Jean Rodgers Jeremy C. Mottram Michael P. Barrett James M. Brewer 《PLoS neglected tropical diseases》2015,9(4)
Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma. 相似文献
78.
79.
Alexandr N. Simonov Jessica K. Holien Joyee Chun In Yeung Ann D. Nguyen C. Jo Corbin Jie Zheng Vladimir L. Kuznetsov Richard J. Auchus Alan J. Conley Alan M. Bond Michael W. Parker Raymond J. Rodgers Lisandra L. Martin 《PloS one》2015,10(11)
Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. 相似文献
80.
Jennifer Hanratty Nuala Livingstone Shannon Robalino Caroline B. Terwee Magdalena Glod Inalegwu P. Oono Jacqui Rodgers Geraldine Macdonald Helen McConachie 《PloS one》2015,10(12)