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  • 1
    Publication Date: 2013-09-06
    Description: The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein-small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and beta-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898436/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898436/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tinberg, Christine E -- Khare, Sagar D -- Dou, Jiayi -- Doyle, Lindsey -- Nelson, Jorgen W -- Schena, Alberto -- Jankowski, Wojciech -- Kalodimos, Charalampos G -- Johnsson, Kai -- Stoddard, Barry L -- Baker, David -- P41 GM103533/GM/NIGMS NIH HHS/ -- R01 GM049857/GM/NIGMS NIH HHS/ -- T32 HG000035/HG/NHGRI NIH HHS/ -- T32 HG00035/HG/NHGRI NIH HHS/ -- England -- Nature. 2013 Sep 12;501(7466):212-6. doi: 10.1038/nature12443. Epub 2013 Sep 4.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24005320" target="_blank"〉PubMed〈/a〉
    Keywords: Binding Sites ; Biotechnology ; *Computer Simulation ; Crystallography, X-Ray ; Digoxigenin/chemistry/*metabolism ; *Drug Design ; Estradiol/chemistry/metabolism ; Ligands ; Models, Molecular ; Progesterone/chemistry/metabolism ; Protein Binding ; Proteins/*chemistry/*metabolism ; Reproducibility of Results ; Substrate Specificity
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
  • 3
    Publication Date: 2015-12-18
    Description: Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures--which is dictated by the internal geometry and local packing of the repeat building blocks--is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed alpha-solenoid repeat structures (alpha-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed alpha-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727831/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727831/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Doyle, Lindsey -- Hallinan, Jazmine -- Bolduc, Jill -- Parmeggiani, Fabio -- Baker, David -- Stoddard, Barry L -- Bradley, Philip -- R01 GM049857/GM/NIGMS NIH HHS/ -- R01 GM115545/GM/NIGMS NIH HHS/ -- R01GM49857/GM/NIGMS NIH HHS/ -- R21 GM106117/GM/NIGMS NIH HHS/ -- R21GM106117/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2015 Dec 24;528(7583):585-8. doi: 10.1038/nature16191. Epub 2015 Dec 16.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Seattle, Washington 98109, USA. ; Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA. ; Institute for Protein Design, University of Washington, Seattle, Washington 98195, USA. ; Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA. ; Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Seattle, Washington 98019, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26675735" target="_blank"〉PubMed〈/a〉
    Keywords: *Amino Acid Motifs ; *Bioengineering ; *Computer Simulation ; Crystallography, X-Ray ; Databases, Protein ; Models, Molecular ; *Protein Structure, Secondary ; Proteins/*chemistry ; Reproducibility of Results
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 2013-04-20
    Description: We present the detection of five planets--Kepler-62b, c, d, e, and f--of size 1.31, 0.54, 1.95, 1.61 and 1.41 Earth radii (R plus sign in circle), orbiting a K2V star at periods of 5.7, 12.4, 18.2, 122.4, and 267.3 days, respectively. The outermost planets, Kepler-62e and -62f, are super-Earth-size (1.25 R plus sign in circle 〈 planet radius 〈/= 2.0 R plus sign in circle) planets in the habitable zone of their host star, respectively receiving 1.2 +/- 0.2 times and 0.41 +/- 0.05 times the solar flux at Earth's orbit. Theoretical models of Kepler-62e and -62f for a stellar age of ~7 billion years suggest that both planets could be solid, either with a rocky composition or composed of mostly solid water in their bulk.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Borucki, William J -- Agol, Eric -- Fressin, Francois -- Kaltenegger, Lisa -- Rowe, Jason -- Isaacson, Howard -- Fischer, Debra -- Batalha, Natalie -- Lissauer, Jack J -- Marcy, Geoffrey W -- Fabrycky, Daniel -- Desert, Jean-Michel -- Bryson, Stephen T -- Barclay, Thomas -- Bastien, Fabienne -- Boss, Alan -- Brugamyer, Erik -- Buchhave, Lars A -- Burke, Chris -- Caldwell, Douglas A -- Carter, Josh -- Charbonneau, David -- Crepp, Justin R -- Christensen-Dalsgaard, Jorgen -- Christiansen, Jessie L -- Ciardi, David -- Cochran, William D -- DeVore, Edna -- Doyle, Laurance -- Dupree, Andrea K -- Endl, Michael -- Everett, Mark E -- Ford, Eric B -- Fortney, Jonathan -- Gautier, Thomas N 3rd -- Geary, John C -- Gould, Alan -- Haas, Michael -- Henze, Christopher -- Howard, Andrew W -- Howell, Steve B -- Huber, Daniel -- Jenkins, Jon M -- Kjeldsen, Hans -- Kolbl, Rea -- Kolodziejczak, Jeffery -- Latham, David W -- Lee, Brian L -- Lopez, Eric -- Mullally, Fergal -- Orosz, Jerome A -- Prsa, Andrej -- Quintana, Elisa V -- Sanchis-Ojeda, Roberto -- Sasselov, Dimitar -- Seader, Shawn -- Shporer, Avi -- Steffen, Jason H -- Still, Martin -- Tenenbaum, Peter -- Thompson, Susan E -- Torres, Guillermo -- Twicken, Joseph D -- Welsh, William F -- Winn, Joshua N -- New York, N.Y. -- Science. 2013 May 3;340(6132):587-90. doi: 10.1126/science.1234702. Epub 2013 Apr 18.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉NASA Ames Research Center, Moffett Field, CA 94035, USA. william.j.borucki@nasa.gov〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23599262" target="_blank"〉PubMed〈/a〉
    Keywords: Exobiology ; Extraterrestrial Environment ; Models, Theoretical ; *Planets ; Stars, Celestial ; *Water
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 5
    Publication Date: 2018-07-19
    Description: Evidence suggests that the α 4 β 2, but not the α 7, subtype of the nicotinic acetylcholine receptor (nAChR) plays a key role in mediating the behavioral effects of nicotine and related drugs. However, the importance of other nAChR subtypes remains unclear. The present studies were conducted to examine the involvement of nAChR subtypes by determining the effects of selected nicotinic agonists and antagonists in squirrel monkeys either 1) responding for food reinforcement or 2) discriminating the nicotinic agonist (+)-epibatidine (0.001 mg/kg) from vehicle. In food-reinforcement studies, nicotine, (+)-epibatidine, varenicline and cytisine all produced dose-dependent decreases in rates of food-maintained responding. The rate-decreasing effects of nicotine were antagonized by mecamylamine (nonselective), not appreciably altered by dihydro- β -erythroidine ( α 4 β 2 selective), and exacerbated by the nicotinic partial agonists, varenicline and cytisine. Results from discrimination studies show that non-nicotinic drugs did not substitute for (+)-epibatidine, and that except for lobeline, the nicotinic agonists produced either full [(+)-epibatidine, (–)-epibatidine, and nicotine] or partial (varenicline, cytisine, anabaseine, and isoarecolone) substitution for (+)-epibatidine. In interaction studies with antagonists differing in selectivity, (+)-epibatidine discrimination was substantively antagonized by mecamylamine, slightly attenuated by hexamethonium (peripherally restricted) or dihydro- β -erythroidine, and not altered by methyllycaconitine ( α 7 selective). Varenicline and cytisine enhanced (+)-epibatidine’s discriminative-stimulus effects. Correlational analysis revealed a close correspondence between relative behavioral potencies of nicotinic agonists in both studies and their published relative binding affinities at α 4 β 2 and α 3 β 4, but not α 7 nAChR, subtypes. Collectively, these results are consistent with the idea that the α 4 β 2 and α 3 β 4, but not α 7 nAChR subtypes play a role in the behavioral effects of nicotinic agonists.
    Print ISSN: 0022-3565
    Electronic ISSN: 1521-0103
    Topics: Medicine
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  • 6
    ISSN: 0032-8332
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Perineal swelling was correlated with changes in vaginal cytology and serum ovarian hormone levels and with development of the ovarian follicle. The average length of 66 menstrual cycles as seen in 12 mature baboons was 35.7±.66 days with a range of 25 to 47 days. Laparoscopic observations and photographic documentation of follicular development were made as early as 13 days prior to ovulation with the most rapid follicular maturation occurring 24 to 48 hours before ovulation. In 38.5% of the cycles ovulation occurred on the last day of maximal perineal tumescence with 26.9% of the ovulations occurring one day after initial detumescence. In 17.8% ovulations occurred two to five days prior to detumescence while the remaining 17.8% occurred two to three days following first observed detumescence. The mean progesterone level during the follicular phase was significantly less than that in the luteal phase, 1.6 and 6.7 ng/ml respectively (p〈0.01). The level of estrogen detected during the periods of maximal tumescence was significantly higher (p〈0.05) than that detected in the remainder of the cycle, 19.8 and 5.9 pg/ml respectively. Vaginal smears were stained, observed microscopically, and found to correlate with perineal swelling, hormone levels, and laparoscopic observations.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    [S.l.] : American Institute of Physics (AIP)
    Journal of Applied Physics 79 (1996), S. 7223-7226 
    ISSN: 1089-7550
    Source: AIP Digital Archive
    Topics: Physics
    Notes: A tunable dye laser has been used to selectively photo-pump transitions in a laser ablated YBa2Cu3O7 plume. The resultant fluorescence and scattered light has been monitored both with a photo-multiplier tube and a gated intensified CCD array camera. The spatial and temporal characteristics of the fluorescence are used to demonstrate that phonons emitted within the plume are strongly reabsorbed, and thus radiation is effectively trapped. Measurement of the degree of trapping is limited due to an alternative radiative decay channel, but a maximum escape probability of about 10% can be inferred, even several centimeters from the target surface, and more than 10 μs after the initial ablation pulse. The implications for plasma spectroscopy and laser induced fluorescence studies of ablated plumes are discussed. © 1996 American Institute of Physics.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1476-5535
    Keywords: Megasphaera cerevisiae ; 16S rRNA ; DNA probe ; beer spoilage ; phylogeny
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The 16S ribosomal RNA gene from the beer-spoilage organism,Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced. Analysis confirmed the phylogenetic position ofM. cerevisiae as a sister taxon ofMegasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci. The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.
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  • 9
    ISSN: 1432-0843
    Keywords: Key words MS-209 ; Reversal drug ; Multidrug resistance ; MRP ; Lung cancer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purpose and methods: MS-209 is a newly synthesized quinoline compound used orally to overcome human P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). The multidrug resistance-associated protein (MRP) gene is thought to play an important role in MDR in lung cancer. To investigate whether MS-209 could also overcome MRP-mediated MDR, we examined the effect of the compound using a cytotoxicity assay on MDR1 gene-negative drug-selected MDR and wildtype lung cancer cells with various levels of MRP gene expression. The effects of MS-209 were compared with those of verapamil (VER) and cyclosporin A (CsA). The level of MRP gene expression in the cells was evaluated semiquantitatively by RT-PCR. For vincristine (VCR), intracellular accumulation of [3H]-VCR was measured with or without MS-209. Results: In MDR UMCC-1/VP small-cell lung carcinoma cell line, 5 μM of MS-209 and VER enhanced the cytotoxicity of etoposide, doxorubicin (DOX) and VCR more than twofold, and completely reversed the resistance to VCR. The mean reversing effects of MS-209 on DOX and VCR were significantly stronger than those of VER and CsA. In wildtype non-small-cell lung carcinoma cells, the effects of MS-209 were almost equal to those of VER and CsA. The effect of these three agents correlated with the level of MRP gene expression. The MS-209-induced increase in intracellular accumulation of VCR was proportional to the level of MRP gene expression in these cells. Conclusion: Our results indicate that MS-209 is a potentially useful drug that can overcome MRP-mediated intrinsic and acquired MDR in human lung cancer.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Altered phosphorus concentration, oxygen supply rate, and programmed addition of sucrose and phosphorus were applied to Dioscorea deltoidea plant cells in airlift suspension to increase the formation of the secondary metabolite diosgenin. A low oxygen supply rate (kla′ of 3.9 h-1) completely inhibited formation of diosgenin. A high oxygen supply rate (kla′ of 17.1 h-1) led to the greatest formation of diosgenin in 30 g/L sucrose when the sucrose-to-phosphorus mole ratio was 42.5:1. Programmed addition of nutrients over a 15-day period reduced growth of cell mass relative to diosgenin mass. Intentional aggregation by entrapment of virtually all cells in reticulated polyurethane foam led to reduced cell-mass yield, diosgenin yield and concentration relative to suspended cells at the same conditions. Entrapment of a small fraction of the cells led to a delayed development of a suspension culture and to formation of significantly higher concentrations of diosgenin. Nearly all of this increase was attributable to the suspended cells. This result suggested sequestering of nonproductive cells by the matrix or sequestering of important nutrients by the matrix-bound cells. Entrapped cells attained densities of 40 g/L in the matrix.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
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