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31.
32.
Stephanie A. Schaefer Ming Dong Renee P. Rubenstein Wayne A. Wilkie Brian J. Bahnson Colin Thorpe Sharon Rozovsky 《Journal of molecular biology》2013,425(2):222-231
Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable 77Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate 77Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, 77Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis. 相似文献
33.
Spores of Ganoderma applanatum were collected by gravity. The spore extract was prepared and its biochemical and immunological properties were examined. In isoelectric focusing, the extract had 10–12 bands with pl values between 3.5 to 6.0, the strongest being at 3.5, 3.75, 4.55 and 5.2. In crossedradioimmunoelectrophoresis using human atopic sera, the extract had two dominant and two minor allergens. In IgE immunoblots of sodium dodecylsulphate polyacrylamide gel electrophoresis, the extract showed reactivity at 82, 45, 33, 26, 16, 11 kDa MW, the strongest being at 45 kDa MW. These results indicate that G. applanatum contains a complex mixture of allergens. 相似文献
34.
Carl R. Illig Carl L. Manthey Sanath K. Meegalla Mark J. Wall Jinsheng Chen Kenneth J. Wilson Renee L. DesJarlais Shelley K. Ballentine Carsten Schubert Carl S. Crysler Yanmin Chen Christopher J. Molloy Margery A. Chaikin Robert R. Donatelli Edward Yurkow Zhao Zhou Mark R. Player Bruce E. Tomczuk 《Bioorganic & medicinal chemistry letters》2013,23(23):6363-6369
Structure–activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC50 0.7 nM, FMS cell IC50 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats. 相似文献
35.
Lehua Deng Polina Tsybina Katie J. Gregg Renee Mosi Wesley F. Zandberg Alisdair B. Boraston David J. Vocadlo 《Bioorganic & medicinal chemistry》2013,21(16):4839-4845
Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors. 相似文献
36.
Christine N. Kay Renee C. Ryals George V. Aslanidi Seok Hong Min Qing Ruan Jingfen Sun Frank M. Dyka Daniel Kasuga Andrea E. Ayala Kim Van Vliet Mavis Agbandje-McKenna William W. Hauswirth Sanford L. Boye Shannon E. Boye 《PloS one》2013,8(4)
Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors. 相似文献
37.
Hussam H. Shaheen Bianka Prinz Ming-Tang Chen Tej Pavoor Song Lin Nga Rewa Houston-Cummings Renee Moore Terrance A. Stadheim Dongxing Zha 《PloS one》2013,8(7)
State-of-the-art monoclonal antibody (mAb) discovery methods that utilize surface display techniques in prokaryotic and eukaryotic cells require multiple steps of reformatting and switching of hosts to transition from display to expression. This results in a separation between antibody affinity maturation and full-length mAb production platforms. Here, we report for the first time, a method in Glyco-engineered
Pichia
pastoris
that enables simultaneous surface display and secretion of full-length mAb molecules with human-like N-glycans using the same yeast cell. This paradigm takes advantage of homo-dimerization of the Fc portion of an IgG molecule to a surface-anchored "bait" Fc, which results in targeting functional “half” IgGs to the cell wall of
Pichia
pastoris
without interfering with the secretion of full length mAb. We show the utility of this method in isolating high affinity, well-expressed anti-PCSK9 leads from a designed library that was created by mating yeasts containing either light chain or heavy chain IgG libraries. Coupled with Glyco-engineered
Pichia
pastoris
, this method provides a powerful tool for the discovery and production of therapeutic human mAbs in the same host thus improving drug developability and potentially shortening the discovery time cycle. 相似文献
38.
Jared M. Baeten Renee Heffron Lara Kidoguchi Nelly R. Mugo Elly Katabira Elizabeth A. Bukusi Stephen Asiimwe Jessica E. Haberer Jennifer Morton Kenneth Ngure Nulu Bulya Josephine Odoyo Edna Tindimwebwa Craig Hendrix Mark A. Marzinke Norma C. Ware Monique A. Wyatt Susan Morrison Harald Haugen Andrew Mujugira Deborah Donnell Connie Celum Partners Demonstration Project Team 《PLoS medicine》2016,13(8)
BackgroundAntiretroviral-based interventions for HIV-1 prevention, including antiretroviral therapy (ART) to reduce the infectiousness of HIV-1 infected persons and pre-exposure prophylaxis (PrEP) to reduce the susceptibility of HIV-1 uninfected persons, showed high efficacy for HIV-1 protection in randomized clinical trials. We conducted a prospective implementation study to understand the feasibility and effectiveness of these interventions in delivery settings.ConclusionsIntegrated delivery of time-limited PrEP until sustained ART use in African HIV-1-serodiscordant couples was feasible, demonstrated high uptake and adherence, and resulted in near elimination of HIV-1 transmission, with an observed HIV incidence of <0.5% per year compared to an expected incidence of >5% per year. 相似文献
39.
Tyler M. Sharp Brenda Rivera García Janice Pérez-Padilla Renee L. Galloway Marta Guerra Kyle R. Ryff Dana Haberling Sharada Ramakrishnan Sean Shadomy Dianna Blau Kay M. Tomashek William A. Bower 《PLoS neglected tropical diseases》2016,10(2)
Background
Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.Methodology/Principal Findings
During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).Conclusions/Significance
Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis. 相似文献40.
Carboxypeptidase D is the only enzyme responsible for antibody C‐terminal lysine cleavage in Chinese hamster ovary (CHO) cells 下载免费PDF全文