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91.
Dimitra Sakoula Garrett J. Smith Jeroen Frank Rob J. Mesman Linnea F. M. Kop Pieter Blom Mike S. M. Jetten Maartje A. H. J. van Kessel Sebastian Lücker 《The ISME journal》2022,16(4):958
The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.Subject terms: Environmental microbiology, Sequencing, Microbiology 相似文献
92.
Takada A Feldmann H Stroeher U Bray M Watanabe S Ito H McGregor M Kawaoka Y 《Journal of virology》2003,77(2):1069-1074
Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals. 相似文献
93.
Discovery and characterization of cooperative ligand binding in the adaptive region of interleukin-2
The cytokine hormone interleukin-2 (IL-2) contains a highly adaptive region that binds small, druglike molecules. The binding properties of this adaptive region have been explored using a "tethering" method that relies on the formation of a disulfide bond between the protein and small-molecule ligands. Using tethering, surface plasmon resonance (SPR), and X-ray crystallography, we have discovered that the IL-2 adaptive region contains at least two cooperative binding sites where the binding of a first ligand to one site promotes or antagonizes the binding of a second ligand to the second site. Cooperative energies of interaction of -2 kcal/mol are observed. The observation that the adaptive region contains two adjacent sites may lead to the development of tight-binding antagonists of a protein-protein interaction. Cooperative ligand binding in the adaptive region of IL-2 underscores the importance of protein dynamics in molecular recognition. The tethering approach provides a novel and general strategy for discovering such cooperative binding interactions in specific, flexible regions of protein structure. 相似文献
94.
Conticello SG Kowalsman ND Jacobsen C Yudkovsky G Sato K Elazar Z Petersen CM Aronheim A Fainzilber M 《The Journal of biological chemistry》2003,278(29):26311-26314
Misfolded secretory proteins are retained in the endoplasmic reticulum (ER) by quality control mechanisms targeted to exposed hydrophobic surfaces. Paradoxically, certain conotoxins expose extensive hydrophobic surfaces upon folding to their bioactive structures. How then can such secreted mini-proteins traverse the secretory pathway? Here we show that secretion of the hydrophobic conotoxin-TxVI is strongly dependent on its propeptide domain, which enhances TxVI export from the ER. The propeptide domain interacts with sorting receptors from the sortilin Vps10p domain family. The sortilin-TxVI interaction occurs in the ER, and sortilin facilitates export of TxVI from the ER to the Golgi. Thus, the prodomain in a secreted hydrophobic protein acts as a tag that can facilitate its ER export by a hitchhiking mechanism. 相似文献
95.
Pütz MM Bouche FB de Swart RL Muller CP 《International journal for parasitology》2003,33(5-6):525-545
Vaccination with the current live attenuated measles vaccine is one of the most successful and cost-effective medical interventions. However, as a result of persisting maternal antibodies and immaturity of the infant immune system, this vaccine is poorly immunogenic in children <9 months old. Immunity against the live vaccine is less robust than natural immunity and protection less durable. There may also be some concern about (vaccine) virus spread during the final stage of an eventual measles eradication program. Opinions may differ with respect to the potential threat that some of these concerns may be to the World Health Organisation goal of measles elimination, but there is a consensus that the development of new measles vaccines cannot wait. Candidate vaccines are based on viral or bacterial vectors expressing recombinant viral proteins, naked DNA, immune stimulating complexes or synthetic peptides mimicking neutralising epitopes. While some of these candidate vaccines have proven their efficacy in monkey studies, aerosol formulated live attenuated measles vaccine are evaluated in clinical trials. 相似文献
96.
97.
Oligomerization of soluble Fas antigen induces its cytotoxicity 总被引:6,自引:0,他引:6
Proussakova OV Rabaya NA Moshnikova AB Telegina ES Turanov A Nanazashvili MG Beletsky IP 《The Journal of biological chemistry》2003,278(38):36236-36241
Soluble Fas antigen can protect cells against Fas-mediated apoptosis. High level soluble Fas antigen characteristic for blood of patients with autoimmune disease or cancer is believed to prevent the elimination of autoimmune lymphocytes or tumor cells. Here we first report that human recombinant FasDeltaTM, i.e. soluble Fas generated by alternative splicing of the intact exon 6, is capable of inducing death of transformed cells by "reverse" apoptotic signaling via transmembrane Fas ligand. FasDeltaTM, as well as transmembrane Fas antigen, can be either monomeric or oligomeric, and both its forms are efficient in blocking Fas-mediated apoptosis, although the cytotoxic activity is exhibited solely by the latter. An in vivo analysis of soluble Fas antigen showed that unlike in healthy controls, nearly the total FasDeltaTM present in sera of rheumatoid arthritis patients was oligomeric. This resulted in suppression of cell proliferation in the experimental sera and in its promotion in controls. Thus, oligomerization/depolymerization of soluble Fas antigen can regulate its activity and contribute to the pathogenesis of autoimmune diseases and cancer. 相似文献
98.
99.
Baker KN Rendall MH Patel A Boyd P Hoare M Freedman RB James DC 《Trends in biotechnology》2002,20(4):149-156
Specific measurement of recombinant protein titer in a complex environment during industrial bioprocessing has traditionally relied on labor-intensive and time-consuming immunoassays. In recent years, however, developments in analytical technology have resulted in improved methods for protein product monitoring during bioprocessing. The choice of product-monitoring technology for a particular bioprocess will depend on a variety of assay factors and instrument-specific factors. In this article, we have compiled an overview of the advantages and disadvantages of the most commonly used technologies used: electrochemiluminescence, optical biosensors, rapid chromatography and nephelometry. The advantages of each technology for measuring both small and large recombinant therapeutic proteins are compared with a conventional enzyme-linked immunosorbent assay (ELISA) technique. 相似文献
100.
We have developed a relational database, called the General Repository for Interaction Datasets (The GRID; ) to archive and display physical, genetic and functional interactions. The GRID displays data-rich interaction tables for any protein of interest, combines literature-derived and high throughput interaction datasets, and is readily accessible via the World Wide Web. Interactions parsed in the GRID can be viewed in graphical form with a versatile visualization tool called Osprey. 相似文献