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151.
Martin Aeschbacher Christoph A. Reinhardt Gerhard Zbinden 《Cell biology and toxicology》1986,2(2):247-255
A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA
dimethyl sulfate
- DMSO
dimethyl sulfoxide
- EB
ethidium bromide
- F
fluorescein
- FDA
fluorescein diacetate
- FS25
concentration of test substance resulting in a F-peak left-shift of 25% from control
- PBS
phosphate buffered saline
- SCT
forward light scatter
- SDS
sodium dodecyl sulfate 相似文献
152.
Stressor‐induced biodiversity gradients: revisiting biodiversity–ecosystem functioning relationships 下载免费PDF全文
Christoph Mensens Frederik De Laender Colin R. Janssen Koen Sabbe Marleen De Troch 《Oikos》2015,124(6):677-684
Biodiversity–ecosystem functioning experiments typically inspect functioning in randomly composed communities, representing broad gradients of taxonomic richness. We tested if the resulting evenness gradients and evenness–functioning relationships reflect those found in communities facing evenness loss caused by anthropogenic stressors. To this end, we exposed marine benthic diatom communities to a series of treatments with the herbicide atrazine, and analysed the relationship between the resulting gradients of evenness and ecosystem functioning (primary production, energy content and sediment stabilization). Atrazine exposure resulted in narrower evenness gradients and steeper evenness–functioning relations than produced by the design of random community assembly. The disproportionately large decrease in functioning following atrazine treatment was related to selective atrazine effects on the species that contributed most to the ecosystem functions considered. Our findings demonstrate that the sensitivity to stress and the contribution to ecosystem functioning at the species level should be both considered to understand biodiversity and ecosystem functioning under anthropogenic stress. Synthesis Biodiversity loss affects ecosystem functioning, yet biodiversity–ecosystem functioning relations have mainly been investigated using communities with random species loss. In nature however, species are lost according to their sensitivity to environmental stress. In the present study, biodiversity loss and biodiversity–ecosystem functioning relations in randomly composed diatom communities were compared to those induced by the pesticide atrazine. Stress exposure resulted in smaller biodiversity loss but steeper decrease in functioning than in randomly composed communities, due to selective atrazine effects on the best performing species. Therefore, species‐specific sensitivity and contribution to ecosystem functioning need to be considered to predict biodiversity and ecosystem functioning under anthropogenic stress. 相似文献
153.
Martin Wagner Damien R. Ashby Caroline Kurtz Ahsan Alam Mark Busbridge Ulrike Raff Josef Zimmermann Peter U. Heuschmann Christoph Wanner Lothar Schramm 《PloS one》2015,10(4)
Background
Anemia is common and is associated with impaired clinical outcomes in diabetic chronic kidney disease (CKD). It may be explained by reduced erythropoietin (EPO) synthesis, but recent data suggest that EPO-resistance and diminished iron availability due to inflammation contribute significantly. In this cohort study, we evaluated the impact of hepcidin-25—the key hormone of iron-metabolism—on clinical outcomes in diabetic patients with CKD along with endogenous EPO levels.Methods
249 diabetic patients with CKD of any stage, excluding end-stage renal disease (ESRD), were enrolled (2003–2005), if they were not on EPO-stimulating agent and iron therapy. Hepcidin-25 levels were measured by radioimmunoassay. The association of hepcidin-25 at baseline with clinical variables was investigated using linear regression models. All-cause mortality and a composite endpoint of CKD progression (ESRD or doubling of serum creatinine) were analyzed by Cox proportional hazards models.Results
Patients (age 67 yrs, 53% male, GFR 51 ml/min, hemoglobin 131 g/L, EPO 13.5 U/L, hepcidin-25 62.0 ng/ml) were followed for a median time of 4.2 yrs. Forty-nine patients died (19.7%) and forty (16.1%) patients reached the composite endpoint. Elevated hepcidin levels were independently associated with higher ferritin-levels, lower EPO-levels and impaired kidney function (all p<0.05). Hepcidin was related to mortality, along with its interaction with EPO, older age, greater proteinuria and elevated CRP (all p<0.05). Hepcidin was also predictive for progression of CKD, aside from baseline GFR, proteinuria, low albumin- and hemoglobin-levels and a history of CVD (all p<0.05).Conclusions
We found hepcidin-25 to be associated with EPO and impaired kidney function in diabetic CKD. Elevated hepcidin-25 and EPO-levels were independent predictors of mortality, while hepcidin-25 was also predictive for progression of CKD. Both hepcidin-25 and EPO may represent important prognostic factors of clinical outcome and have the potential to further define “high risk” populations in CKD. 相似文献154.
E. Fernando Cagua Jesse E. M. Cochran Christoph A. Rohner Clare E. M. Prebble Tane H. Sinclair-Taylor Simon J. Pierce Michael L. Berumen 《Biology letters》2015,11(4)
Although whale sharks (Rhincodon typus) have been documented to move thousands of kilometres, they are most frequently observed at a few predictable seasonal aggregation sites. The absence of sharks at the surface during visual surveys has led to the assumption that sharks disperse to places unknown during the long ‘off-seasons’ at most of these locations. Here we compare 2 years of R. typus visual sighting records from Mafia Island in Tanzania to concurrent acoustic telemetry of tagged individuals. Sightings revealed a clear seasonal pattern with a peak between October and February and no sharks observed at other times. By contrast, acoustic telemetry demonstrated year-round residency of R. typus. The sharks use a different habitat in the off-season, swimming deeper and further away from shore, presumably in response to prey distributions. This behavioural change reduces the sharks'' visibility, giving the false impression that they have left the area. We demonstrate, for the first time to our knowledge, year-round residency of unprovisioned, individual R. typus at an aggregation site, and highlight the importance of using multiple techniques to study the movement ecology of marine megafauna. 相似文献
155.
Frank?L?hr Franz?Tumulka Christoph?Bock Rupert?AbeleEmail author Volker?D?tschEmail author 《Journal of biomolecular NMR》2015,62(3):263-279
Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in 1H–15N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins. 相似文献
156.
Marco Dollinger René Müller-Wille Florian Zeman Michael Haimerl Christoph Niessen Lukas P. Beyer Sven A. Lang Andreas Teufel Christian Stroszczynski Philipp Wiggermann 《PloS one》2015,10(8)
Purpose
To evaluate risk factors associated with alterations in venous structures adjacent to an ablation zone after percutaneous irreversible electroporation (IRE) of hepatic malignancies at subacute follow-up (1 to 3 days after IRE) and to describe evolution of these alterations at mid-term follow-up.Materials and Methods
43 patients (men/women, 32/11; mean age, 60.3 years) were identified in whom venous structures were located within a perimeter of 1.0 cm of the ablation zone at subacute follow-up after IRE of 84 hepatic lesions (primary/secondary hepatic tumors, 31/53). These vessels were retrospectively evaluated by means of pre-interventional and post-interventional contrast-enhanced magnetic resonance imaging or computed tomography or both. Any vascular changes in flow, patency, and diameter were documented. Correlations between vascular change (yes/no) and characteristics of patients, lesions, and ablation procedures were assessed by generalized linear models.Results
191 venous structures were located within a perimeter of 1.0 cm of the ablation zone: 55 (29%) were encased by the ablation zone, 78 (41%) abutted the ablation zone, and 58 (30%) were located between 0.1 and 1.0 cm from the border of the ablation zone. At subacute follow-up, vascular changes were found in 19 of the 191 vessels (9.9%), with partial portal vein thrombosis in 2, complete portal vein thrombosis in 3, and lumen narrowing in 14 of 19. At follow-up of patients with subacute vessel alterations (mean, 5.7 months; range, 0 to 14 months) thrombosis had resolved in 2 of 5 cases; vessel narrowing had completely resolved in 8 of 14 cases, and partly resolved in 1 of 14 cases. The encasement of a vessel by ablation zone (OR = 6.36, p<0.001), ablation zone being adjacent to a portal vein (OR = 8.94, p<0.001), and the usage of more than 3 IRE probes (OR = 3.60, p = 0.035) were independently associated with post-IRE vessel alterations.Conclusion
Venous structures located in close proximity to an IRE ablation zone remain largely unaffected by this procedure, and thrombosis is rare. 相似文献157.
Mitcheell Maestre‐Martínez Katja Haupt Frank Edlich Piotr Neumann Christoph Parthier Milton T. Stubbs Gunter Fischer Christian Lücke 《Journal of molecular recognition : JMR》2011,24(1):23-34
The Bcl‐2 inhibitor FKBP38 is regulated by the Ca2+‐sensor calmodulin (CaM). Here we show a hitherto unknown low‐affinity cation‐binding site in the FKBP domain of FKBP38, which may afford an additional level of regulation based on electrostatic interactions. Fluorescence titration experiments indicate that in particular the physiologically relevant Ca2+ ion binds to this site. NMR‐based chemical shift perturbation data locate this cation‐interaction site within the β5–α1 loop (Leu90–Ile96) of the FKBP domain, which contains the acidic Asp92 and Asp94 side‐chains. Binding constants were subsequently determined for K+, Mg2+, Ca2+, and La3+, indicating that the net charge and the radius of the ion influences the binding interaction. X‐ray diffraction data furthermore show that the conformation of the β5–α1 loop is influenced by the presence of a positively charged guanidinium group belonging to a neighboring FKBP38 molecule in the crystal lattice. The position of the cation‐binding site has been further elucidated based on pseudocontact shift data obtained by NMR via titration with Tb3+. Elimination of the Ca2+‐binding capacity by substitution of the respective aspartate residues in a D92N/D94N double‐substituted variant reduces the Bcl‐2 affinity of the FKBP3835–153/CaM complex to the same degree as the presence of Ca2+ in the wild‐type protein. Hence, this charge‐sensitive site in the FKBP domain participates in the regulation of FKBP38 function by enabling electrostatic interactions with ligand proteins and/or salt ions such as Ca2+. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
158.
159.
Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels. 相似文献
160.
Katina I Spanier Florian Leese Christoph Mayer John K Colbourne Don Gilbert Michael E Pfrender Ralph Tollrian 《BMC molecular biology》2010,11(1):50