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51.
R Graf R Mattera J Codina M K Estes L Birnbaumer 《The Journal of biological chemistry》1992,267(34):24307-24314
The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide. 相似文献
52.
53.
Integration of cell line and process development to overcome the challenge of a difficult to express protein 下载免费PDF全文
Swati Dalvi Bryan St. Germain Wenqi Xie Scott Estes Rashmi Kshirsagar Thomas Ryll 《Biotechnology progress》2015,31(5):1201-1211
This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi‐prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench‐top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10‐fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1201–1211, 2015 相似文献
54.
Margarida RG Maia Lal C Chaudhary Charles S Bestwick Anthony J Richardson Nest McKain Tony R Larson Ian A Graham Robert J Wallace 《BMC microbiology》2010,10(1):52
Background
Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA. 相似文献55.
Engineered antifouling microtopographies - effect of feature size, geometry, and roughness on settlement of zoospores of the green alga Ulva 总被引:2,自引:0,他引:2
Schumacher JF Carman ML Estes TG Feinberg AW Wilson LH Callow ME Callow JA Finlay JA Brennan AB 《Biofouling》2007,23(1-2):55-62
The effect of feature size, geometry, and roughness on the settlement of zoospores of the ship fouling alga Ulva was evaluated using engineered microtopographies in polydimethylsiloxane elastomer. The topographies studied were designed at a feature spacing of 2 microm and all significantly reduced spore settlement compared to a smooth surface. An indirect correlation between spore settlement and a newly described engineered roughness index (ERI) was identified. ERI is a dimensionless ratio based on Wenzel's roughness factor, depressed surface fraction, and the degree of freedom of spore movement. Uniform surfaces of either 2 mum diameter circular pillars (ERI=5.0) or 2 microm wide ridges (ERI=6.1) reduced settlement by 36% and 31%, respectively. A novel multi-feature topography consisting of 2 mum diameter circular pillars and 10 microm equilateral triangles (ERI=8.7) reduced spore settlement by 58%. The largest reduction in spore settlement, 77%, was obtained with the Sharklet AF topography (ERI=9.5). 相似文献
56.
An NSP4-dependant mechanism by which rotavirus impairs lactase enzymatic activity in brush border of human enterocyte-like Caco-2 cells 总被引:1,自引:0,他引:1
Beau I Cotte-Laffitte J Géniteau-Legendre M Estes MK Servin AL 《Cellular microbiology》2007,9(9):2254-2266
Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23-62) is a brush border membrane (BBM)-associated enzyme in intestinal cells that hydrolyse lactose, the most important sugar in milk. Impairing in lactase activity during rotavirus infection has been described in diseased infants but the mechanism by which the functional lesion occurs remains unknown. We undertook a study to elucidate whether rotavirus impairs the lactase enzymatic activity in BBM of human enterocyte cells. In this study we use cultured human intestinal fully differentiated enterocyte-like Caco-2 cells to demonstrate how the lactase enzymatic activity at BBM is significantly decreased in rhesus monkey rotavirus (RRV)-infected cells. We found that the decrease in enzyme activity is not dependent of the Ca(2+)- and cAMP-dependent signalling events triggered by the virus. The LPH biosynthesis, stability, and expression of the protein at the BBM of infected cells were not modified. We provide evidence that in RRV-infected cells the kinetic of lactase enzymatic activity present at the BBM was modified. Both BBM(control) and BBM(RRV) have identical K(m) values, but hydrolyse the substrate at different rates. Thus, the BBM(RRV) exhibits almost a 1.5-fold decreased V(max) than that of BBM(control) and is therefore enzymatically less active than the latter. Our study demonstrate conclusively that the impairment of lactase enzymatic activity at the BBM of the enterocyte-like Caco-2 cells observed during rotavirus infection results from an inhibitory action of the secreted non-structural rotavirus protein NSP4. 相似文献
57.
P. J. Faria G. D. Kavembe J. O. Jung’a C. N. Kimwele L. D. Estes P. R. Reillo A. G. Mwangi M. W. Bruford 《Conservation Genetics》2011,12(3):745-751
The mountain bongo antelope Tragelaphus eurycerus isaaci has rapidly declined in recent decades, due to a combination of hunting, habitat degradation and disease. Endemic to Kenya,
mountain bongo populations have shrunk to approximately 100 individuals now mainly confined to the Aberdares mountain ranges.
Indirect observation of bongo signs (e.g. tracks, dung) can be misleading, thus methods to ensure reliable species identification,
such as DNA-based techniques, are necessary to effectively study and monitor this species. We assessed bongo presence in four
mountain habitats in Kenya (Mount Kenya National Park, Aberdare National Park, Eburu and Mau forests) and carried out a preliminary
analysis of genetic variation by examining 466 bp of the first domain of the mtDNA control region using DNA extracted from
faecal samples. Of the 201 dung samples collected in the field, 102 samples were molecularly identified as bongo, 97 as waterbuck,
one as African buffalo and one as Aders’ duiker. Overall species-identification accuracy by experienced trackers was 64%,
with very high error of commission when identifying bongo sign (37%), and high error of omission for waterbuck sign (82%),
suggesting that the two species’ signs are easily confused. Despite high variation in the mtDNA control region in most antelope
species, our results suggest low genetic variation in mountain bongo as only two haplotypes were detected in 102 samples analyzed.
In contrast, the analysis of 63 waterbuck samples from the same sites revealed 21 haplotypes. Nevertheless, further examination
using nuclear DNA markers (e.g. microsatellites) in a multi-locus approach is still required, especially because the use of
mitochondrial DNA can result in population overestimation as distinct dung samples can potentially be originated from the
same individual. 相似文献
58.
McCann MD Lim GF Manni ML Estes J Klapec KA Frattini GD Knarr RJ Gratton JL Serra MJ 《RNA (New York, N.Y.)》2011,17(1):108-119
Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop. 相似文献
59.
Suzanne Estes Anna L Coleman-Hulbert Kiley A Hicks Gene de Haan Sarah R Martha Jeremiah B Knapp Samson W Smith Kevin C Stein Dee R Denver 《BMC evolutionary biology》2011,11(1):11
Background
Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism. 相似文献60.
ANDREA GHIRARDO WERNER HELLER MATTHIAS FLADUNG JÖRG‐PETER SCHNITZLER HILKE SCHROEDER 《Plant, cell & environment》2012,35(12):2192-2207
The indirect defences of plants are comprised of herbivore‐induced plant volatiles (HIPVs) that among other things attract the natural enemies of insects. However, the actual extent of the benefits of HIPV emissions in complex co‐evolved plant‐herbivore systems is only poorly understood. The observation that a few Quercus robur L. trees constantly tolerated (T‐oaks) infestation by a major pest of oaks (Tortrix viridana L.), compared with heavily defoliated trees (susceptible: S‐oaks), lead us to a combined biochemical and behavioural study. We used these evidently different phenotypes to analyse whether the resistance of T‐oaks to the herbivore was dependent on the amount and scent of HIPVs and/or differences in non‐volatile polyphenolic leaf constituents (as quercetin‐, kaempferol‐ and flavonol glycosides). In addition to non‐volatile metabolic differences, typically defensive HIPV emissions differed between S‐oaks and T‐oaks. Female moths were attracted by the blend of HIPVs from S‐oaks, showing significantly higher amounts of (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT) and (E)‐β‐ocimene and avoid T‐oaks with relative high fraction of the sesquiterpenes α‐farnesene and germacrene D. Hence, the strategy of T‐oaks exhibiting directly herbivore‐repellent HIPV emissions instead of high emissions of predator‐attracting HIPVs of the S‐oaks appears to be the better mechanism for avoiding defoliation. 相似文献