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41.
Modulating the function of the measles virus RNA-dependent RNA polymerase by insertion of green fluorescent protein into the open reading frame
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Measles virus (MV) is the type species of the Morbillivirus genus and its RNA-dependent RNA polymerase complex is comprised of two viral polypeptides, the large (L) and the phospho- (P) proteins. Sequence alignments of morbillivirus L polymerases have demonstrated the existence of three well-conserved domains (D1, D2, and D3) which are linked by two variable hinges (H1 and H2). Epitope tags (c-Myc) were introduced into H1 and H2 to investigate the tolerance of the variable regions to insertions and to probe the flexibility of the proposed domain structures to spatial reorientation. Insertion into H1 abolished polymerase activity whereas introduction into H2 had no effect. The open reading frame of enhanced green fluorescent protein was also inserted into the H2 region of the MV L gene to extend these observations. This resulted in a recombinant protein that was both functional and autofluorescent, although the overall polymerase activity was reduced by over 40%. Two recombinant viruses which contained the chimeric L genes EdtagL(MMc-mycM) and EdtagL(MMEGFPM) were generated. Tagged L proteins were detectable, by indirect immunofluorescence in the case of EdtagL(MMc-mycM) and by autofluorescence in the case of EdtagL(MMEGFPM). We suggest that D3 enjoys a limited conformational independence from the other domains, indicating that the L polymerases of the Mononegavirales may function as multidomain proteins. 相似文献
42.
43.
Morrissey JP Walsh UF O'Donnell A Moënne-Loccoz Y O'Gara F 《Antonie van Leeuwenhoek》2002,81(1-4):599-606
The major growth seen in the biotechnology industry in recent decades has largely been driven by the exploitation of genetic
engineering techniques. The initial benefits have been predominantly in the biomedical area, with products such as vaccines
and hormones that have received broad public approval. In the environmental biotechnology and industrial ecology sectors,
biotechnology has the potential to make significant advances through the use of genetically modified (GM) microbial inoculants
that can reduce agri-chemical usage or remediate polluted environments. Although many GM inoculants have been developed and
tested under laboratory conditions, commercial exploitation has lagged behind. Here, we review scientific and regulatory requirements
that must be satisfied as part of that exploitation process. Particular attention is paid to new European Union (EU) regulations
(Directives) that govern the testing and release of genetically modified organisms and microbial plant protection inoculants
in the EU. With regard to the release of GM inoculants, the impact of the inoculant and the fate of modified genes are important
concerns. Long term monitoring of release sites is necessary to address these issues. Data are reported from the monitoring
of a site 6 years after release of GM Sinorhizobium meliloti strains. It was found that despite the absence of a host plant, the GM strains persisted in the soil for at least 6 years.
Horizontal transfer and microevolution of a GM plasmid between S. meliloti strains was also observed. These data illustrate the importance of assessing the long-term persistence of GM inoculants.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
44.
Federica Lombardi Kalyan Golla Darren J. Fitzpatrick Fergal P. Casey Niamh Moran Denis C. Shields 《PLoS computational biology》2015,11(4)
Identifying effective therapeutic drug combinations that modulate complex
signaling pathways in platelets is central to the advancement of effective
anti-thrombotic therapies. However, there is no systems model of the platelet
that predicts responses to different inhibitor combinations. We developed an
approach which goes beyond current inhibitor-inhibitor combination screening to
efficiently consider other signaling aspects that may give insights into the
behaviour of the platelet as a system. We investigated combinations of platelet
inhibitors and activators. We evaluated three distinct strands of information,
namely: activator-inhibitor combination screens (testing a panel of inhibitors
against a panel of activators); inhibitor-inhibitor synergy screens; and
activator-activator synergy screens. We demonstrated how these analyses may be
efficiently performed, both experimentally and computationally, to identify
particular combinations of most interest. Robust tests of activator-activator
synergy and of inhibitor-inhibitor synergy required combinations to show
significant excesses over the double doses of each component. Modeling
identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and
complementarity between inhibitor-inhibitor synergy effects and
activator-inhibitor combination effects. This approach accelerates the mapping
of combination effects of compounds to develop combinations that may be
therapeutically beneficial. We integrated the three information sources into a
unified model that predicted the benefits of a triple drug combination targeting
ADP, thromboxane and thrombin signaling. 相似文献
45.
46.
Nina Luckgei Birgit Habenstein Francesco Ravotti Simon Megy Francois Penin Jean-Baptiste Marchand Fergal Hill Anja Böckmann Beat H. Meier 《Biomolecular NMR assignments》2014,8(1):1-6
The complement 4 binding protein (C4bp) plays a crucial role in the inhibition of the complement cascade. It has an extraordinary seven-arm octopus-like structure with 7 tentacle-like identical chains, held together at their C-terminal end. The C-terminal domain does oligomerize in isolation, and is necessary and sufficient to oligomerize full-length C4bp. It is predicted to form a seven-helix coiled coil, and its multimerization properties make it a promising vaccine adjuvant, probably by enhancing the structural stability and binding affinity of the presented antigen. Here, we present the solid-state NMR resonance assignment of the human C4bp C-terminal oligomerization Domain, hC4pbOD, and the corresponding secondary chemical shifts. 相似文献
47.
In tissue engineering, bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. This study examined the effect of short term flow perfusion bioreactor culture, prior to long‐term static culture, on human osteoblast cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold for bone tissue engineering. Human fetal osteoblasts (hFOB 1.19) were seeded onto CG scaffolds and pre‐cultured for 6 days. Constructs were then placed into the bioreactor and exposed to 3 × 1 h bouts of steady flow (1 mL/min) separated by 7 h of no flow over a 24‐h period. The constructs were then cultured under static osteogenic conditions for up to 28 days. Results show that the bioreactor and static culture control groups displayed similar cell numbers and metabolic activity. Histologically, however, peripheral cell‐encapsulation was observed in the static controls, whereas, improved migration and homogenous cell distribution was seen in the bioreactor groups. Gene expression analysis showed that all osteogenic markers investigated displayed greater levels of expression in the bioreactor groups compared to static controls. While static groups showed increased mineral deposition; mechanical testing revealed that there was no difference in the compressive modulus between bioreactor and static groups. In conclusion, a flow perfusion bioreactor improved construct homogeneity by preventing peripheral encapsulation whilst also providing an enhanced osteogenic phenotype over static controls. Bioeng. 2011; 108:1203–1210. © 2010 Wiley Periodicals, Inc. 相似文献
48.
Biopharmaceuticals manufacturing is a critical component of the modern healthcare system, with emerging new treatments composed of increasingly complex biomolecules offering solutions to chronic and debilitating disorders. While this sector continues to grow, it strongly exhibits “boom-to-bust” performance which threatens its long-term viability. Future trends within the industry indicate a shift towards continuous production systems using single use technologies that raises sustainability issues, yet research in this area is sparse and lacks consideration of the complex interactions between environmental, social and economic concerns. The authors outline a sustainability-focused vision and propose opportunities for research to aid the development of a more integrated approach that would enhance the sustainability of the industry. 相似文献
49.
Emily K. Forbes Simone C. de Cassan David Llewellyn Sumi Biswas Anna L. Goodman Matthew G. Cottingham Carole A. Long Richard J. Pleass Adrian V. S. Hill Fergal Hill Simon J. Draper 《PloS one》2012,7(9)
Viral vectored vaccines have been shown to induce both T cell and antibody responses in animals and humans. However, the induction of even higher level T cell responses may be crucial in achieving vaccine efficacy against difficult disease targets, especially in humans. Here we investigate the oligomerization domain of the α-chain of C4b-binding protein (C4 bp) as a candidate T cell “molecular adjuvant” when fused to malaria antigens expressed by human adenovirus serotype 5 (AdHu5) vectored vaccines in BALB/c mice. We demonstrate that i) C-terminal fusion of an oligomerization domain can enhance the quantity of antigen-specific CD4+ and CD8+ T cell responses induced in mice after only a single immunization of recombinant AdHu5, and that the T cells maintain similar functional cytokine profiles; ii) an adjuvant effect is observed for AdHu5 vectors expressing either the 42 kDa C-terminal domain of Plasmodium yoelii merozoite surface protein 1 (PyMSP142) or the 83 kDa ectodomain of P. falciparum strain 3D7 apical membrane antigen 1 (PfAMA1), but not a candidate 128kDa P. falciparum MSP1 biallelic fusion antigen; iii) following two homologous immunizations of AdHu5 vaccines, antigen-specific T cell responses are further enhanced, however, in both BALB/c mice and New Zealand White rabbits no enhancement of functional antibody responses is observed; and iv) that the T cell adjuvant activity of C4 bp is not dependent on a functional Fc-receptor γ-chain in the host, but is associated with the oligomerization of small (<80 kDa) antigens expressed by recombinant AdHu5. The oligomerization domain of C4 bp can thus adjuvant T cell responses induced by AdHu5 vectors against selected antigens and its clinical utility as well as mechanism of action warrant further investigation. 相似文献
50.
R Bukowski RT Chlebowski I Thune AS Furberg GD Hankins FD Malone ME D'Alton 《PloS one》2012,7(7):e40199