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1.
Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins 总被引:4,自引:0,他引:4
Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation. 相似文献
2.
By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected. SDS-PAGE analysis of mutant culture supernatant samples showed production of a 38–40 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on three peptides the protein in the mutant culture was identified as CPO. No differences in the CPO gene sequences of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and mutant were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38–40 kDa was detected. Our results reveal that the mutant protein lacks the heme group as well as the N-glycans. 相似文献
3.
In our on‐going pursuit to discover natural products and natural product‐based compounds to control the bacterial species Flavobacterium columnare, which causes columnaris disease in channel catfish (Ictalurus punctatus), we synthesized flavone and chalcone analogs, and evaluated these compounds, along with flavonoids from natural sources, for their antibacterial activities against two isolates of F. columnare (ALM‐00‐173 and BioMed) using a rapid bioassay. The flavonoids chrysin ( 1a ), 5,7‐dihydroxy‐4′‐methoxyflavone ( 11 ), isorhamnetin ( 26 ), luteolin ( 27 ), and biochanin A ( 29 ), and chalcone derivative 8b showed strong antibacterial activities against F. columnare ALM‐00‐173 based on minimum inhibition concentration (MIC) results. Flavonoids 1a, 8, 11, 13 (5,4′‐dihydroxy‐7‐methoxyflavone), 26 , and 29 exhibited strong antibacterial activities against F. columnare BioMed based upon MIC results. The 24‐h 50% inhibition concentration (IC50) results revealed that 27 and 29 were the most active compounds against F. columnare ALM‐00‐173 (IC50 of 7.5 and 8.5 mg/l, resp.), while 26 and 29 were the most toxic compound against F. columnare BioMed (IC50 of 9.2 and 3.5 mg/l, resp.). These IC50 results were lower than those obtained for wogonin against F. columnare ALM‐00‐173 and F. columnare BioMed (28.4 and 5.4 mg/l, resp.). However, based on MIC results, none of the compounds evaluated in this study were as active as wogonin (MIC 0.3 mg/l for each F. columnare isolate). Further modification of the wogonin structure to enhance antibacterial is of interest. 相似文献
4.
Hay DC Pernagallo S Diaz-Mochon JJ Medine CN Greenhough S Hannoun Z Schrader J Black JR Fletcher J Dalgetty D Thompson AI Newsome PN Forbes SJ Ross JA Bradley M Iredale JP 《Stem cell research》2011,6(2):92-102
Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of "hepatocytes"; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources. 相似文献
5.
Schramm DD Karim M Schrader HR Holt RR Kirkpatrick NJ Polagruto JA Ensunsa JL Schmitz HH Keen CL 《Life sciences》2003,73(7):857-869
Macronutrients in food and gastric acid are known to have a pronounced effect on the metabolism of many xenobiotics, an effect that impacts their efficacy as bioactive agents. In this investigation we assessed the impact of select food treatments and the histamine H(2)-receptor antagonist Famotidine (Pepcid-AC) on flavanol absorption and metabolism. Four crossover intervention studies were conducted with 6 subjects each. Volunteers consumed sugar-free, flavanol-rich cocoa (0.125 g/kg body wt) alone, with macronutrient-rich foods (8.75 or 17.5 kJ/kg subject body wt) or Famotidine (Pepcid-AC). Blood samples were drawn at 5 time points including baseline. Plasma samples were analyzed for epicatechin and catechin flavanols by HPLC. Pharmacokinetic parameters were assessed using non-compartmental methodology. When provided at 17.5 kJ/kg subject body weight (approximately 4 kcal/kg), sugar and bread test meals increased flavanol area under the curve (AUC) values to 140% of control values (P < 0.05). A corresponding tendency for plasma antioxidant capacity to increase was observed for the cocoa treatment at 1.5 and 2.5 h (P < 0.17, P < 0.06, respectively). The ability of treatment meals to affect AUC values was positively correlated with treatment carbohydrate content (r = 0.83; P< 0.02). In contrast to carbohydrate rich meals, lipid and protein rich meals and Famotidine treatment had minimal effects on flavanol absorption. Based on C(max) and AUC values, this data suggests that the uptake of flavanols can be increased significantly by concurrent carbohydrate consumption. 相似文献
6.
Vallabh O. Shah Casey Carroll Ryan Mals Donica Ghahate Jeanette Bobelu Phillip Sandy Kathleen Colleran Ronald Schrader Thomas Faber Mark R. Burge 《PloS one》2015,10(5)
Introduction
One in three people will be diagnosed with diabetes by 2050, and the proportion will likely be higher among Native Americans. Diabetes control is currently suboptimal in underserved populations despite a plethora of new therapies. Patient empowerment is a key determinant of diabetes control, but such empowerment can be difficult to achieve due to resource limitation and cultural, language and health literacy barriers. We describe a home-based educational intervention using Community Health Representatives (CHRs), leading to improvement in Patient Activation Measures scores and clinical indicators of diabetes control.Methods
Sixty participants with type 2 diabetes (T2D) completed a baseline evaluation including physical exam, Point of Care (POC) testing, and the Patient Activation Measure (PAM) survey. Participants then underwent a one hour group didactic session led by Community Health Representatives (CHRs) who subsequently carried out monthly home-based educational interventions to encourage healthy lifestyles, including diet, exercise, and alcohol and cigarette avoidance until follow up at 6 months, when clinical phenotyping and the PAM survey were repeated.Results
PAM scores were increased by at least one level in 35 (58%) participants, while 24 participants who started at higher baseline score did not change. Six months after intervention, mean levels of A1C decreased by 0.7 ± 1.2%; fasting blood glucose decreased by 24.0 ± 38.0 mg/dl; BMI decreased by 1.5 ± 2.1 kg/m2; total cholesterol decreased by 12.0± 28.0 mg/dl; and triglycerides decreased by 52.0 ± 71.0 mg/dl. All of these changes were statistically significant (p<0.05).Conclusion
This six month, CHR led and community-oriented educational intervention helps inform standards of practice for the management of diabetes, engages diabetic populations in their own care, and reduces health disparities for the underserved population of Zuni Indians.Trial Registration
ClinicalTrials.gov NCT02339311 相似文献7.
Zhi L Ringgenberg JD Edwards JP Tegley CM West SJ Pio B Motamedi M Jones TK Marschke KB Mais DE Schrader WT 《Bioorganic & medicinal chemistry letters》2003,13(12):2075-2078
A series of 1,2-dihydrochromeno[3,4-f]quinoline derivatives was synthesized and tested in biological assays to evaluate the nonsteroidal progesterone receptor modulator pharmacophore (4) as antiprogestins. A number of potent analogues were identified by modification of the substituents at the D-ring. 相似文献
8.
The use of plant functional traits has become increasingly popular in ecological studies because plant functional traits help to understand key ecological processes in plant species and communities. This also includes changes in diversity, inter‐ and intraspecific interactions, and relationships of species at different spatiotemporal scales. Leaf traits are among the most important traits as they describe key dimensions of a plant's life history strategy. Further, leaf area is a key parameter with relevance for other traits such as specific leaf area, which in turn correlates with leaf chemical composition, photosynthetic rate, leaf longevity, and carbon investment. Measuring leaf area usually involves the use of scanners and commercial software and can be difficult under field conditions. We present Leaf‐IT, a new smartphone application for measuring leaf area and other trait‐related areas. Leaf‐IT is free, designed for scientific purposes, and runs on Android 4 or higher. We tested the precision and accuracy using objects with standardized area and compared the area measurements of real leaves with the well‐established, commercial software WinFOLIA using the Altman–Bland method. Area measurements of standardized objects show that Leaf‐IT measures area with high accuracy and precision. Area measurements with Leaf‐IT of real leaves are comparable to those of WinFOLIA. Leaf‐IT is an easy‐to‐use application running on a wide range of smartphones. That increases the portability and use of Leaf‐IT and makes it possible to measure leaf area under field conditions typical for remote locations. Its high accuracy and precision are similar to WinFOLIA. Currently, its main limitation is margin detection of damaged leaves or complex leaf morphologies. 相似文献
9.
Kaderali L Zander T Faigle U Wolf J Schultze JL Schrader R 《Bioinformatics (Oxford, England)》2006,22(12):1495-1502
MOTIVATION: DNA microarrays allow the simultaneous measurement of thousands of gene expression levels in any given patient sample. Gene expression data have been shown to correlate with survival in several cancers, however, analysis of the data is difficult, since typically at most a few hundred patients are available, resulting in severely underdetermined regression or classification models. Several approaches exist to classify patients in different risk classes, however, relatively little has been done with respect to the prediction of actual survival times. We introduce CASPAR, a novel method to predict true survival times for the individual patient based on microarray measurements. CASPAR is based on a multivariate Cox regression model that is embedded in a Bayesian framework. A hierarchical prior distribution on the regression parameters is specifically designed to deal with high dimensionality (large number of genes) and low sample size settings, that are typical for microarray measurements. This enables CASPAR to automatically select small, most informative subsets of genes for prediction. RESULTS: Validity of the method is demonstrated on two publicly available datasets on diffuse large B-cell lymphoma (DLBCL) and on adenocarcinoma of the lung. The method successfully identifies long and short survivors, with high sensitivity and specificity. We compare our method with two alternative methods from the literature, demonstrating superior results of our approach. In addition, we show that CASPAR can further refine predictions made using clinical scoring systems such as the International Prognostic Index (IPI) for DLBCL and clinical staging for lung cancer, thus providing an additional tool for the clinician. An analysis of the genes identified confirms previously published results, and furthermore, new candidate genes correlated with survival are identified. 相似文献
10.
Amphiphysins interact directly with clathrin and have a function in clathrin-mediated synaptic vesicle recycling and clathrin-mediated endocytosis. The neuronal isoform amphiphysin-1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Rephosphorylation was stimulated by nerve growth factor. We analysed the regulation of amphiphysin-clathrin interactions by phosphorylation. The N-terminal domain of clathrin bound to unphosphorylated amphiphysin-1, but not to the phosphorylated protein. A search for possible phosphorylation sites revealed two casein kinase 2 consensus motifs in close proximity to the clathrin binding sites in amphiphysin-1 and -2. We mutagenized these residues (T350 and T387) to glutamate, mimicking a constitutive phosphorylation. The double mutant showed a strong reduction in clathrin binding. The assumption that casein kinase 2 phosphorylates amphiphysin-1 at T350 and T387 was corroborated by experiments showing that: (i) casein kinase 2 phosphorylated these residues directly in vitro, (ii) when expressed in HeLa cells, the glutamate mutant showed reduced phosphorylation, and (iii) casein kinase 2 inhibitors blocked nerve growth factor-induced phosphorylation of endogenous amphiphysin-1 in PC12 cells. These observations are consistent with the hypothesis that, upon activation by nerve growth factor, casein kinase 2 phosphorylates amphiphysin-1 and thereby regulates the endocytosis of clathrin-coated vesicles via the interaction between clathrin and amphiphysin. 相似文献