Lanthanide Spectroscopic Studies of the Dinuclear and Mg(II)-Dependent PvuII Restriction Endonuclease

Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10−20 μM), Eu(III) excitation 7F0 → 5D0 spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 μM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (Kd = 1−3 μM) and a much weaker site (Kd ≈ 100−200 μM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 μM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function

Development and validation of the brief esophageal dysphagia questionnaire

BackgroundEsophageal dysphagia is common in gastroenterology practice and has multiple etiologies. A complication for some patients with dysphagia is food impaction. A valid and reliable questionnaire to rapidly evaluate esophageal dysphagia and impaction symptoms can aid the gastroenterologist in gathering information to inform treatment approach and further evaluation, including endoscopy.Methods1638 patients participated over two study phases. 744 participants completed the Brief Esophageal Dysphagia Questionnaire (BEDQ) for phase 1; 869 completed the BEDQ, Visceral Sensitivity Index, Gastroesophageal Reflux Disease Questionnaire, and Hospital Anxiety and Depression Scale for phase 2. Demographic and clinical data were obtained via the electronic medical record. The BEDQ was evaluated for internal consistency, split‐half reliability, ceiling and floor effects, and construct validity.Key ResultsThe BEDQ demonstrated excellent internal consistency, reliability, and construct validity. The symptom frequency and severity scales scored above the standard acceptable cutoffs for reliability while the impaction subscale yielded poor internal consistency and split‐half reliability; thus the impaction items were deemed qualifiers only and removed from the total score. No significant ceiling or floor effects were found with the exception of 1 item, and inter‐item correlations fell within accepted ranges. Construct validity was supported by moderate yet significant correlations with other measures. The predictive ability of the BEDQ was small but significant.Conclusions & InferencesThe BEDQ represents a rapid, reliable, and valid assessment tool for esophageal dysphagia with food impaction for clinical practice that differentiates between patients with major motor dysfunction and mechanical obstruction.Validated, rapid clinical assessment tools for esophageal dysphagia are lacking. The brief esophageal dysphagia questionnaire aims to gauge the severity and frequency of dysphagia with additional items to gauge food impaction. The BEDQ is a reliable and valid tool to assess esophageal dysphagia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135130/1/nmo12889.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135130/2/nmo12889_am.pd

Moisture analysis of an extratropical cyclone

Summary in German.Includes bibliographical references (pages 297-298)

Prevalence of Diarrhea and Enteropathogens in Racing Sled Dogs

Background: Diarrhea is highly prevalent in racing sled dogs, although the underlying causes are poorly understood. Hypothesis: Clostridium perfringens enterotoxin (CPE) and Clostridium difficile Toxin A and B are associated with diarrhea in racing sled dogs. Animals: One hundred and thirty-five sled dogs. Methods: Freshly voided feces were obtained from 55 dogs before racing and from 80 dogs after 400 miles of racing. Samples were visually scored for diarrhea, mucus, blood, and melena. CPE and C. difficile Toxin A and B were detected by ELISA. Samples were cultured for C. perfringens, C. difficile, Campylobacter, Salmonella, and Escherichia coli 0157; Giardia and Cryptosporidium spp. were detected via immunofluorescence. Results: Diarrhea occurred in 36% of dogs during racing, and hematochezia, fecal mucus or melena, or all 3 occurred in 57.5% of dogs. Salmonella was isolated from 78.2% of dogs before racing, and from 71.3% of dogs during racing. C. perfringens and C. difficile were isolated from 100 and 58.2% of dogs before racing, and from 95 and 36.3% of dogs during racing. Dogs were more likely to test positive for CPE during than before racing (18.8 versus 5.5%, P 5 .021); however, no enteropathogens or their respective toxins were significantly associated with hematochezia or diarrhea. Conclusions and Clinical Importance: Sled dogs participating in long distance racing have a high prevalence of diarrhea and hematochezia that is not associated with common enteropathogens. It is possible that diarrhea and hematochezia represent the effect of prolonged exercise on the gastrointestinal tract

Carbon stable isotope analysis of cereal remains as a way to reconstruct water availability: preliminary results

Reconstructing past water availability, both as rainfall and irrigation, is important to answer questions about the way society reacts to climate and its changes and the role of irrigation in the development of social complexity. Carbon stable isotope analysis of archaeobotanical remains is a potentially valuable method for reconstructing water availability. To further define the relationship between water availability and plant carbon isotope composition and to set up baseline values for the Southern Levant, grains of experimentally grown barley and sorghum were studied. The cereal crops were grown at three stations under five different irrigation regimes in Jordan. Results indicate that a positive but weak relationship exists between irrigation regime and total water input of barley grains, but no relationship was found for sorghum. The relationship for barley is site-specific and inter-annual variation was present at Deir ‘Alla, but not at Ramtha and Khirbet as-Samra

The Lantern Vol. 39, No. 2, Spring 1973

• Days of Rain • Reflections On Clifton, New Jersey • Interlude • Window Scene • Eh! • Odyssey of Malcolm • Tuna on Toast • The Second Avenue Bus • Salutation of the Dawn • So Say Something • Mood • Moriarty\u27s Lament • I\u27ve Been a Lonely Gypsy • Change • Cool Ray • The Thinker • A Southern Sunsethttps://digitalcommons.ursinus.edu/lantern/1102/thumbnail.jp

Signatures of arithmetic simplicity in metabolic network architecture

Metabolic networks perform some of the most fundamental functions in living cells, including energy transduction and building block biosynthesis. While these are the best characterized networks in living systems, understanding their evolutionary history and complex wiring constitutes one of the most fascinating open questions in biology, intimately related to the enigma of life's origin itself. Is the evolution of metabolism subject to general principles, beyond the unpredictable accumulation of multiple historical accidents? Here we search for such principles by applying to an artificial chemical universe some of the methodologies developed for the study of genome scale models of cellular metabolism. In particular, we use metabolic flux constraint-based models to exhaustively search for artificial chemistry pathways that can optimally perform an array of elementary metabolic functions. Despite the simplicity of the model employed, we find that the ensuing pathways display a surprisingly rich set of properties, including the existence of autocatalytic cycles and hierarchical modules, the appearance of universally preferable metabolites and reactions, and a logarithmic trend of pathway length as a function of input/output molecule size. Some of these properties can be derived analytically, borrowing methods previously used in cryptography. In addition, by mapping biochemical networks onto a simplified carbon atom reaction backbone, we find that several of the properties predicted by the artificial chemistry model hold for real metabolic networks. These findings suggest that optimality principles and arithmetic simplicity might lie beneath some aspects of biochemical complexity

Structural shape optimization using Cartesian grids and automatic h-adaptive mesh projection

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