Tritium exchange reactions catalyzed by 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli K-12.

Tritiated water and tritiated substrates have been used to study exchange reactions catalyzed by Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16, 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate). With pyruvate, the enzyme catalyzes a rapid first-order exchange of all three methyl hydrogens in the absence of added acceptor aldehyde (i.e. glyoxylate). This reaction is not rate limiting for aldol condensation or cleavage; quite different pH-activity profiles for the exchange reaction versus aldol cleavage and also comparative effects that pH changes have on Km and V values for the two processes favor this conclusion. The exchange reaction with 2-oxobutyrate, a substrate analog, is stereoselective; one methylene hydrogen is removed at a 6-fold faster rate than the other but eventually both are exchanged. No tritium exchange occurs with glyoxylate.     

Purification,properties, and mode of action of hemicellulase I produced by Ceratocystis paradoxa

A culture isolate (CP₂) of the fungal plant pathogen Ceratocystis paradoxa produces at least five extra-cellular hemicellulases when grown on a medium containing a commercial hemicellulose as inducer. One of the five enzymes, hemicellulase I (HC-I), was purified by ammonium sulphate precipitation, ion-exchange chromatography (DEAE-Sephadex and then Cellex-CM), and iso-electric focusing at pH 3–10 and 8–10. HC-I behaves as a single protein on electrophoresis at pH 6.0 and 8.4. The enzyme degrades hemicellulose B (an arabino-4-O-methylglucuronoxylan) and arabinoxylan to arabinose, xylose, xylobiose (Xyl₂; β-D-Xylp-(1→4)-D-Xyl), and a mixture of arabinose-xylose and xylose oligosaccharides (AraXyl_n and Xyl_n, where n  3, 4, or 5). The enzyme is deduced to be an endo-enzyme. Xylotetraose (Xyl₄) was the lowest homologue of the xylose oligosaccharides attacked, yielding xylobiose and xylotriose (Xyl₃) only. A mechanism is postulated for this reaction. AraXyl₂AraXyl₅ were slowly hydrolysed to arabinose and the respective xylose saccharide (Xyl₂Xyl₅), and thence to Xyl₂ and Xyl₃. Hydrolysis of the arabinofuranosyl linkage probably does not occur at the same active site as for the xylose oligosaccharides. Hemicellulose B fractions from different sources appeared to be degraded by HC-I. The enzyme showed optimum activity at pH 5.5 and 40°, and K_m was 4.24 mg of hemicellulose/ml.     

Structures of the oligosaccharides from the enzymic hydrolysis of hemicellulose by a hemicellulase of Ceratocystis paradoxa.

And endo-hemicellulase (HC-II) of Ceratocystis paradoxa degraded spear-grass hemicellulose B to a series of mixed oligosaccharides. Four neutral oligosaccharides (AraXyl2, AraXyl3, Xyl2, and Xyl3), isolated by preparative paper chromatography, were shown by enzymic and methylation techniques to constitute a series of beta-(1 leats to 4)-D-xylose and O-alpha-L-arabinofuranosyl-(1 leads to 3)-O-beta-D-xylopyranosyl-(1 leads to 4)-O-beta-D-xylopyranosyl-(1 leads to 4)-D-xylose, respectively, the latter being a new compound.     

Anticipating an unwanted future: euthanasia and dementia in the Netherlands

This ethnographic exploration of anticipation draws on fieldwork among people with dementia and their families in the Netherlands. I examine how requests for euthanasia by people with dementia offer insight into the work of anticipation, revealing it to be a temporal orientation through which the future is made tangible. Imagining a future with dementia may prompt some people to request euthanasia, but timing such measures is extremely difficult and often results in deferral. Contributing to an emerging anthropology of time, I argue that anticipation is a process of establishing, collapsing, and renegotiating the temporal distance between present and future, bringing the future into the present while also, and simultaneously, keeping the future at bay as a continuous ‘not yet’.     

IMPROV-ED trial: eHealth programme for faster recovery and reduced healthcare utilisation after CABG

Netherlands Heart Journal - After coronary artery bypass grafting (CABG), healthcare utilisation is high and is partly unplanned. eHealth applications have been proposed to reduce healthcare...     

Better survival after transcatheter aortic valve replacement by process improvements

ObjectiveThe aim of this study is to assess the effects on procedural, 30-day, and 1‑year all-cause mortality by a newly introduced quality improvement strategy in patients after transcatheter aortic valve replacement (TAVR).MethodsIn October 2015, a coherent set of quality improving interventions with respect to patient geriatric screening, general diagnostic examination and safety of the procedure was implemented at a single centre in the Netherlands. Patients undergoing TAVR in 2013–2018 were included for retrospective analysis. Mortality was assessed in the pre-quality improvement strategy cohort (January 2013 to October 2015; cohort A) and in the post-quality improvement strategy cohort (November 2015 to December 2018; cohort B). Logistic regression analysis was used to estimate the influence of patient and procedural characteristics on the results of the quality improvement strategy in terms of procedural, 30-day, and 1‑year all-cause mortality.ResultsIn total, 806 patients were analysed with 274 patients in cohort A and 532 patients in cohort B. After introduction of the quality improvement strategy, procedural (4.4% to 1.3%, p < 0.01), 30-day (8.4% to 2.7%, p < 0.01) and 1‑year (16.4% to 8.5%, p < 0.01) all-cause mortality significantly decreased. Multivariate regression analysis showed that the quality improvement strategy also significantly reduced 30-day (odds ratio [OR] 0.19, 95% confidence interval [CI] 0.09–0.42) and 1‑year (OR 0.38, 95% CI 0.24–0.61) all-cause mortality if corrected for patient characteristics.ConclusionStructural meetings on evaluation of outcomes highlight potential areas for improvement and subsequent outcome-based quality improvement initiatives can result in lower procedural, 30-day, and 1‑year all-cause mortality.Electronic supplementary materialThe online version of this article (10.1007/s12471-020-01526-7) contains supplementary material, which is available to authorized users.     

Serum proteomics in amnestic mild cognitive impairment

We have explored proteins related to mild cognitive impairment (MCI). The serum proteome of 35 amnestic MCI patients and 35 cognitively healthy persons was investigated by LC MS. We identified 108 differentially expressed peptides between MCI patients and controls, belonging to 39 proteins. Eight proteins were selected for further investigation by quantitative protein measurements using a MRM assay; apolipoprotein E, carboxypeptidase N subunit 2, complement factor B (CFAB), galectin‐3 binding protein (LG3BP), lumican, serum amyloid A‐4 protein (SAA4), serum amyloid P‐component, and sex hormone binding globulin. Results of the quantitative protein measurements showed significantly decreased levels of carboxypeptidase N subunit 2, CFAB, LG3BP, SAA4, and serum amyloid P‐component in serum from amnestic MCI patients compared with cognitive healthy controls (two‐sided t‐test; p < 0.05). Apolipoprotein E and lumican showed no significant difference in protein levels, sex hormone binding globulin could not be quantified since the MRM assay did not reach the required sensitivity. A model based on the three most significantly decreased proteins (CFAB, LG3BP, and SAA4) showed a sensitivity and specificity of 73 and 66%, respectively, for the initial sample set. A small external validation set yielded 77% sensitivity and 75% specificity.     

Collagen Peptides in Urine: A New Promising Biomarker for the Detection of Colorectal Liver Metastases

Introduction

For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls.

Materials and Methods

Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM''s) were developed on a UPLC-MS/MS system.

Results

Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%.

Conclusion

Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.     

Protein interactions between surface annexin A2 and S100A10 mediate adhesion of breast cancer cells to microvascular endothelial cells

Annexin A2 (AnxA2) and S100A10 are known to form a molecular complex. Using fluorescence-based binding assays, we show that both proteins are localised on the cell surface, in a molecular form that allows mutual interaction. We hypothesized that binding between these proteins could facilitate cell–cell interactions. For cells that express surface S100A10 and surface annexin A2, cell–cell interactions can be blocked by competing with the interaction between these proteins. Thus an annexin A2-S100A10 molecular bridge participates in cell–cell interactions, revealing a hitherto unexplored function of this protein interaction.     

Fatty Acid Ethyl Esters Induce Intestinal Epithelial Barrier Dysfunction via a Reactive Oxygen Species-Dependent Mechanism in a Three-Dimensional Cell Culture Model

Background & Aims

Evidence is accumulating that ethanol and its oxidative metabolite, acetaldehyde, can disrupt intestinal epithelial integrity, an important factor contributing to ethanol-induced liver injury. However, ethanol can also be metabolized non-oxidatively generating phosphatidylethanol and fatty acid ethyl esters (FAEEs). This study aims to investigate the effects of FAEEs on barrier function, and to explore the role of oxidative stress as possible mechanism.

Methods

Epithelial permeability was assessed by paracellular flux of fluorescein isothiocyanate-conjugated dextran using live cell imaging. Cell integrity was evaluated by lactate dehydrogenase release. Localization and protein levels of ZO-1 and occludin were analyzed by immunofluorescence and cell-based ELISA, respectively. Intracellular oxidative stress and cellular ATP levels were measured by dichlorofluorescein and luciferase driven bioluminescence, respectively.

Results

In vitro, ethyl oleate and ethyl palmitate dose dependently increased permeability associated with disruption and decreased ZO-1 and occludin protein levels, respectively, and increased intracellular oxidative stress without compromising cell viability. These effects could partially be attenuated by pretreatment with the antioxidant, resveratrol, pointing to the role of oxidative stress in the FAEEs-induced intestinal barrier dysfunction.

Conclusions

These findings show that FAEEs can induce intestinal barrier dysfunction by disrupting the tight junctions, most likely via reactive oxygen species-dependent mechanism.