SKP1 is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an oligosaccharide ...containing Fuc and Gal, which is unusual for a cytoplasmic or nuclear protein. To establish how it is glycosylated, SKP1 labeled with 3HFuc was purified to homogeneity and digested with endo-Lys-C. A single radioactive peptide was found after two-dimensional high performance liquid chromatography. Analysis in a quadrupole time-of-flight mass spectrometer revealed a predominant ion with a novel mass. Tandem mass spectrometry analysis yielded a set of daughter ions which identified the peptide and showed that it was modified at Pro-143. A second series of daughter ions showed that Pro-143 was hydroxylated and derivatized with a potentially linear pentasaccharide, Hex-->Hex-->Fuc-->Hex-->HexNAc-->(HyPro). The attachment site was confirmed by Edman degradation. Gas chromatography-mass spectrometry analysis of trimethylsilyl-derivatives of overexpressed SKP1 after methanolysis showed the HexNAc to be GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1 and from a fucosylation mutant, followed by matrix-assisted laser desorption time-of-flight mass spectrometry analysis, showed that the sugar chain consisted of D-Galpalpha1-->6-D-Galpalpha1-->L-Fucpalpha1-->2-D- Galpbeta1--> 3GlcNAc. Matrix-assisted laser-desorption time-of-flight mass spectrometry analysis of all SKP1 peptides resolved by reversed phase-high performance liquid chromatography showed that SKP1 was only partially hydroxylated at Pro-143 and that all hydroxylated SKP1 was completely glycosylated. Thus SKP1 is variably modified by an unusual linear pentasaccharide, suggesting the localization of a novel glycosylation pathway in the cytoplasm.
Early Eocene fossil floras from British Columbia are a rich resource for reconstructing western North American early Cenozoic climate. The best known of these floras reflect cooler (MAT ≤ 15 °C) ...upland forest communities in contrast to coeval (MAT ≥ 18 °C) forests in lowland western North American sites. Of particular interest is whether Early Eocene climates were monsoonal (highly seasonal precipitation). The McAbee site is a 52.9 ± 0.83 Ma 0.5 km outcrop of bedded lacustrine shale interbedded with volcanic ash. In this report two historical megaflora collections that were collected independently from different stratigraphic levels and (or) laterally separated by ∼100–200 m in the 1980s (University of Saskatchewan) and 2000s (Brandon University) are investigated to (i) assess whether they represent the same leaf population, (ii) assess whether a combined collection yields more precise climate estimates, and (iii) reconstruct paleoclimate to assess the character of regional Early Eocene precipitation seasonality. Combined, the two samples yielded 43 dicot leaf morphotypes. Analysis of leaf size distribution using ANOVA showed no difference between the two samples, and thus they were combined for climate analysis. Climate analysis using leaf physiognomy agrees with previous estimates for McAbee and other regional megafloras, indicating a warm (MAT ∼8–13 °C), mild (CMMT ∼5 °C), moist (MAP > 100 cm/year) ever-wet, non-monsoonal climate. Additionally, we recommend that climate analyses derived from leaf fossils should be based on samples collected within a stratigraphically constrained quarry area to capture a snapshot of climate in time rather than time-averaged estimates derived from multiple quarry sites representing different stratigraphic levels within a fossil site.
Skp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a ...pentasaccharide with the type I blood group H antigen (Fucalpha1,2Galbeta1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have beta-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 beta-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits beta-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo.
Research objectives: This dissertation examines the state of development of each of the eight core electronic health record (EHR) functionalities as described by the IOM and describes how the current ...state of these functionalities limit quality improvement efforts in ambulatory care settings. There is a great deal of literature describing both the potential of the EHR to improve quality of care and showing a lack of improvement associated with EHR use. This study examines the role that the state of development of EHR functionalities plays in the quality improvement. Study design: A qualitative study of four community health center (CHC) networks that provide EHR services to members and three CHCs from each network. Each network used different, commonly used and CCHIT certified EHRs. Sixty five hours of interviews were transcribed, coded, and analyzed from seventy five semi-structured interviews of leaders/staff. The analysis focused on the eight core EHR functionalities as identified by the IOM. Principal findings: Out-of-the-box, none of the EHRs studied strongly supported the provision of guideline based care to individual patients or the management of populations of patients. Extensive EHR modification was needed, with some EHRs requiring more work. Challenges were most acutely felt with templates, interfaces, decision support, and reporting functionalities. Limitations were found less often in administrative processes and within practice messaging. Though EHR functionalities greatly improved based on network and CHC development efforts, focus on quality improvement activities was diminished by the consumption of scarce resources to fix poorly functioning software. Conclusions: Given that EHR adoption rates will continue to increase it should be emphasized that successful QI efforts are difficult to achieve with the current state of the technology, especially for smaller practices. So far the onus of improving the functionalities for use in QI efforts has primarily been left to the EHR adopters, who generally lack the resources to develop the software. Policy needs to take this into account and fund not only EHR implementation, but also ensure great improvements are made to core functionalities. The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.
We introduce FELICIA (FEderated LearnIng with a CentralIzed Adversary) a generative mechanism enabling collaborative learning. In particular, we show how a data owner with limited and biased data ...could benefit from other data owners while keeping data from all the sources private. This is a common scenario in medical image analysis where privacy legislation prevents data from being shared outside local premises. FELICIA works for a large family of Generative Adversarial Networks (GAN) architectures including vanilla and conditional GANs as demonstrated in this work. We show that by using the FELICIA mechanism, a data owner with limited image samples can generate high-quality synthetic images with high utility while neither data owners has to provide access to its data. The sharing happens solely through a central discriminator that has access limited to synthetic data. Here, utility is defined as classification performance on a real test set. We demonstrate these benefits on several realistic healthcare scenarions using benchmark image datasets (MNIST, CIFAR-10) as well as on medical images for the task of skin lesion classification. With multiple experiments, we show that even in the worst cases, combining FELICIA with real data gracefully achieves performance on par with real data while most results significantly improves the utility.
Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of ...Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.
We have used effective reaction rates (ERR) for the helium burning reactions to predict the yield of the gamma-emitting nuclei 26Al, 44Ti, and 60Fe in core col- lapse supernovae. The variations in ...the predicted yields for values of the reaction rates allowed by the ERR are much smaller than obtained previously, and smaller than other uncertainties. A "filter" for supernova nucleosynthesis yields based on pre-supernova structure was used to estimate the effect of failed supernovae on the initial mass function-averaged yields; this substantially reduced the yields of all these isotopes, but the predicted yield ratio 60Fe/26Al was little affected. The robustness of this ratio is promising for comparison with data, but it is larger than observed in nature; possible causes for this discrepancy are discussed.
Below band gap formation of solvated electrons in neutral water clusters using pump-probe photoelectron imaging is compared with recent data for liquid water and with above band gap excitation ...studies in the liquid and clusters. Similar relaxation times in the order of 200 fs and 1-2 ps are retrieved for below and above band gap excitation, in both clusters and liquid. The relaxation times independence from the generation process indicates that these times are dominated by the solvent response, which is significantly slower than the different solvated electron formation processes. The analysis of the temporal evolution of the vertical electron binding energy and the electron binding energy at half maximum suggests a dependence of the solvation time on the binding energy.
Thiol‐affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS‐PAGE and 2D‐PAGE in conjunction ...with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devized to label substrate protectable protein with the thiol specific reagent 14CN‐ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol‐affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by 14CNEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross‐reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol‐affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.
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