We have established an in vitro reconstitution/splicing complementation system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of spliceosome formation. U1 snRNPs reconstituted from purified, native snRNP proteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing-activity to U1-depleted extracts. In vitro reconstituted U1 snRNPs possessing an m3G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function. However, the presence of an m7GpppG or GpppG cap was deleterious to splicing, most likely due to competition for the m7G cap binding proteins. No significant reduction in splicing or E complex formation was detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II, respectively). Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for the U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes. Studies with recombinant U1-C protein mutants indicated that the N-terminal domain of U1-C is necessary and sufficient for the stimulation of E complex formation.
Vitamin D is a fat-soluble hormone that traditionally has been linked to bone health. Recently, its involvement has been extended to other (extra-skeletal) disease areas, such as cancer, CVD, energy metabolism and autoimmune diseases. Vitamin D deficiency is a worldwide problem, and several recommendation-setting bodies have published guidelines for adequate vitamin D intake and status. However, recommendations from, for example, the Health Council of the Netherlands do not provide advice on how to treat vitamin D deficiency, a condition that is often encountered in the clinic. In addition, these recommendations provide guidelines for the maintenance of ‘minimum levels’, and do not advise on ‘optimum levels’ of vitamin D intake/status to further improve health. The NutriProfiel project, a collaboration between the Gelderse Vallei Hospital (Ede, the Netherlands) and the Division of Human Nutrition of Wageningen University (Wageningen, the Netherlands), was initiated to formulate a protocol for the treatment of vitamin deficiency and for the maintenance of optimal vitamin D status. To discuss the controversies around treatment of deficiency and optimal vitamin D status and intakes, a workshop meeting was organised with clinicians, scientists and dietitians. In addition, a literature review was conducted to collect recent information on optimal intake of vitamins, their optimal circulating concentrations, and effective dosing regimens to treat deficiency. This information has been translated into the NutriProfiel advice, which is outlined in this article.
CD64 is a high-affinity FcγRI receptor expressed by activated neutrophils that has been recently evaluated as a potential sepsis parameter. In the present study, the kinetics of neutrophil membrane CD64 expression were examined during a standardized inflammatory response, using a human endotoxemia model, and compared with hematological indices, CRP, cytokines and interleukins. Ten healthy subjects received 2 ng/kg intravenous Escherichia coli lipopolysaccharide (LPS). After administration of LPS, neutrophil CD64 showed a biphasic response, characterized by a first increase from 108.5 ± 7.5 to 133 ± 6 AFU after 1 h ( P = 0.047) and a second increase that started at 6 h and reached its maximum of 167 ± 13 AFU at 22 h ( P < 0.0001). CRP concentrations increased to 40 ± 5 mg/dl 22 h after the administration of LPS. The cytokines and interleukins reached their maximum response within 1—2 h. The maximum values of pro-inflammatory cytokines (TNF-α, IFN-γ and IL-6) correlated with the CD64 expression at 22 h after LPS administration ( r2 = 0.76, r2 = 0.78, r2 = 0.81, respectively, all P < 0.05), whereas this correlation was not found for the anti-inflammatory IL-10 ( r2 = 0.058, P = 0.54), suggesting that neutrophil CD64 expression might be a quantitative marker for innate immunity that could easily be used in the clinical setting.
Procalcitonin (PCT) is a 13-kDa peptide and a precursor of calcitonin. In a healthy population, PCT concentrations are negligible (1 ). In systemic bacterial and fungal infections, plasma concentrations are raised, whereas concentrations remain fairly low in infections of viral or nonspecific cause (2 ). Recent studies have demonstrated the potential of PCT as a parameter to guide antibiotic therapy in different groups of patients, i.e., patients with chronic obstructive pulmonary disease experiencing respiratory tract infections (3,4 ). The most frequently used medical decision points at which the use of antibiotic therapy is considered are 0.25 g/L and 0.50 g/L, depending on the patient population (3, 4 ).The first PCT assays were based on manual immunochemistry methods (Brahms PCT LIA). These assays have been replaced by fully automated immunochemistry methods (Brahms Kryptor, Brahms LIAISON, Olympus SphereLight 180). Recently, the PCT assay has been modified for use on a consolidated routine immunochemistry analyzer family, the Roche Elecsys, cobas, and the Roche Modular E170 systems. We evaluated the analytical performance of this new assay by following the EP10 protocol, a document from the Clinical and Laboratory Standards Institute to test precision, linearity, recovery, carryover, and drift. Samples were prepared at different concentrations, from 0.24 -2.85 g/L. Three aliquots of each concentration were assayed on 5 different days, in a specific assay order. The within-run CV ranged from 3.0% for the lowest concentration to 1.3% for the highest concentration. The between-day CV ranged from 6.3% for the lowest concentration to 2.8% for the highest. These levels of imprecision were comparable with those reported for the PCT assay on the Brahms Kryptor (5 ). The mean recovery was 99%. There was no evidence of nonlinearity or sample carryover. The limit of quantification, i.e., the lowest concentration of analyte that can be quantified with a between-run imprecision of Ͻ20%, met the manufacturer's specification of 0.06 g/L. In addition, we compared the new PCT assay from Roche on the Modular 170 with the widely accepted PCT assay from Brahms on the Kryptor (5 ). For analytical comparison, we used 229 samples of patient serum obtained from 195 different patients who were admitted to our hospital for lower respiratory tract infections (81, exacerbation of chronic obstructive pulmonary disease; 114, pneumonia). The patients participated in an ongoing study in our hospital on the etiology of exacerbations of chronic obstructive pulmonary disease, a study approved by the local ethics committee. Samples were also collected from 34 patients after antibiotic treatment. The majority of the serum samples were obtained within 24 h of admission. Samples not immediately analyzed were stored at Ϫ80°C until analysis. PCT concentrations ranged from 0.02 g/L (limit of detection, i.e., the lowest concentration of analyte that can be reliably measured as being qualitatively present in the sample) to 57 g/L. PCT concentrations...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.