Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Impacts on Micro- and Macro-Structure of Thermally Stabilised Whey Protein-Pectin Complexes: A Fluorescence Approach

Stability of whey protein-pectin complexes is an essential criterion for their application in different food matrices. The impact of process parameters on micro- and macro-structural characteristics of thermally stabilised whey protein-pectin complexes was investigated using fluorescence spectroscopy, ζ-potential measurements, dynamic light scattering and phase separation. Complexes prepared from whey protein isolate (WPI) and pectins with different degrees of esterification (HMP, LMP) were generated at different biopolymer concentrations (WPI + pectin: 5.0 % + 1.0 %, c _{h i g h} ; 2.75 % + 0.55 %, c _{m e d} ; 0.5 % + 0.1 %, c _{l o w} ), heating temperatures (80-90^°C) and pH levels (6.1-4.0). Micro- and macro-structural characteristics of the complexes depended on concentration level and degree of esterification, with complexes being more sensitive towards environmental changes at c _{l o w} than at c _{m e d} and c _{h i g h} . WPI-LMP complexes exhibited sizes <1 μm suitable for micro-encapsulation, whereas WPI-HMP complexes at c _{m e d} achieved sizes from 1-10 μm and at c _{h i g h} from 10-200 μm underlining their potential as fat-replacers and structuring agents, respectively. Slopes and intercepts derived from intensity ratios of fluorescence spectra gave insights into the state of unfolding of β-lactoglobulin within the complexes and thus about the protective effect of pectin addition.     

Self-assembly and photocatalytic activity of branched silicatein/silintaphin filaments decorated with silicatein-synthesized TiO<Subscript>2</Subscript> nanoparticles

The fundamental mechanisms of biomineralization and their translation into innovative synthetic approaches have yielded promising perspectives for the fabrication of biomimetic and bioinspired organic–inorganic hybrid materials. In siliceous sponges, the enzyme silicatein catalyzes the polycondensation of molecular precursors to nano-structured SiO₂ that is deposited on self-assembled filaments consisting of the two silicatein isoforms (silicatein-α and -β) and the scaffold protein silintaphin-1. Due to its broad substrate specificity silicatein is also able to convert in vitro various other precursors to non-biogenic materials (e.g., hydrolysis of titanium bis(ammonium lactato)-dihydroxide [TiBALDH] and subsequent polycondensation to titania [TiO₂]). In the present approach, silicatein was bioengineered to carry a protein tag (Arg-tag) that confers binding affinity to TiO₂. Then, by combining Arg-tagged silicatein-α with silicatein-β and silintaphin-1, self-assembled branched hybrid protein microfilaments were fabricated. Upon subsequent incubation with TiBALDH the filaments were decorated with TiO₂ and assayed for photocatalytic activity through photodegradation of the dye methylene blue. This is the first approach that considers concomitant application of two silicatein isoforms for the synthesis of bioinspired organic–inorganic hybrid materials. It is also the first time that the biocatalytic activity of the enzymes has been combined with both the structure-providing properties of silintaphin-1 and a TiO₂ affinity protein tag to fabricate self-assembled branched protein filaments as template for a silicatein-synthesized TiO₂ photocatalyst. The TiO₂-decorated filaments might be explored as a practical alternative to approaches where biotemplates have to be laboriously isolated from their original biological source prior to TiO₂ immobilization.     

Combinatorial treatment with anacardic acid followed by TRAIL augments induction of apoptosis in TRAIL resistant cancer cells by the regulation of p53, MAPK and NFκβ pathways

TRAIL, an apoptosis inducing cytokine currently in phase II clinical trial, was investigated for its capability to induce apoptosis in six different human tumor cell lines out of which three cell lines showed resistance to TRAIL induced apoptosis. To investigate whether Anacardic acid (A1) an active component of Anacardium occidentale can sensitize the resistant cell lines to TRAIL induced apoptosis, we treated the resistant cells with suboptimal concentration of A1 and showed that it is a potent enhancer of TRAIL induced apoptosis which up-regulates the expression of both DR4 and DR5 receptors, which has been observed in the cellular, protein and mRNA levels. The death receptors upregulation consequent to A1 treatment was corroborated by the activation of p53 as well as phosphorylation of p38 and JNK MAP kinases and concomitant inactivation of NFκβ and ERK signaling cascades. Also, A1 modulated the expression of key apoptotic players like Bax, Bcl-2 and CAD along with the abatement of tumor angiogenesis in vivo in EAT mouse model. Thus, post A1 treatment the TRAIL resistant cells turned into TRAIL sensitive cells. Hence our results demonstrate that A1 can synergize TRAIL induced apoptosis through the upregulation of death receptors and downregulation of anti-apoptotic proteins in cancer context.     

Asymmetric carabid beetle spillover between calcareous grasslands and coniferous forests

Central European calcareous grasslands are considered biodiversity hotspots, but are severely threatened by the change in land-use and by habitat fragmentation. Coniferous forests are typical adjacent habitats to calcareous grasslands, as abandoned calcareous grasslands are often afforested or develop into coniferous forests by succession. To investigate spillover between calcareous grasslands and coniferous forests, a total of 144 pitfall traps for carabid beetles were placed at three different distances (1, 5, 20 m) from the edge in both habitats at eight locations from April to late August. We found that both habitats had a distinct species assemblage and a decrease in spillover with increasing distance from the habitat edge into the adjacent habitat. Calcareous grasslands were more affected by spillover from the adjacent coniferous forests than vice versa because more forest specialists penetrated into calcareous grasslands than grassland specialists penetrated into coniferous forests. We conclude that spillover into small and isolated habitats can severely change species assemblages, which has to be considered in conservation measures. The protection of large sites with small edge-interior ratios can reduce negative effects on species assemblages in endangered calcareous grasslands.     

Spectrofluorimetric determination of dopamine receptor antagonist LE 300 in mice plasma

A simple, rapid and highly sensitive spectrofluorimetric method was developed for determination of a novel type of dopamine receptor antagonist LE300 in mouse plasma. The method is based on measuring the native fluorescence of LE‐300 in methanol at 343 nm after excitation at 280 nm. The fluorescence concentration plot was rectilinear over the range of 3.5–100 ng/mL with a lower detection limit of 1.0 ng/mL and quantification limit of 3.5 ng/mL. The method was statistically validated for linearity, accuracy, precision and selectivity according to the International Conference on Harmonization guidelines. The accuracy and precision results was expressed as % recovery and relative standard deviation (RSD). The accuracy for LE‐300 was in the range 95.5–103.6% and RSD values were in the range of 0.21–1.55% of the theoretical value. The method was successfully applied to the analysis of LE‐300 in mice plasma. The results were compared statistically with those obtained by the reported method and were found to be in good agreement, which could be applied in a pharmacokinetic study. Copyright © 2015 John Wiley & Sons, Ltd.     

Transfemoral Aortic Valve Implantation with the New Edwards Sapien 3 Valve for Treatment of Severe Aortic Stenosis—Impact of Valve Size in a Single Center Experience

Aims

The third generation Edwards Sapien 3 (Edwards Lifesciences Inc., Irvine, California) system was optimized to reduce residual aortic regurgitation and vascular complications.

Methods and Results

235 patients with severe symptomatic aortic stenosis were prospectively enrolled. Transcatheter aortic valve implantations (TAVI) were performed without general anesthesia by transfemoral approach. Patients were followed for 30 days. Patients received 23mm (N = 77), 26mm (N = 91) or 29mm (N = 67) valve based on pre-procedural 256 multislice computer tomography. Mean oversizing did not differ between the 3 valves. There was no residual moderate or severe aortic regurgitation. Rate of mild aortic regurgitation and regurgitation index did not differ between groups. There was no switch to general anesthesia or conversion to surgery. Rate of major vascular complication was 3.0% with no difference between valve and delivery sheath sizes. Within 30 days rates of all cause mortality (2.6%) and stroke (2.1%) were low.

Conclusions

In patients with severe aortic stenosis transfemoral TAVI with the Edwards Sapien 3 valve without general anesthesia was associated with a high rate of device success, no moderate or severe residual aortic regurgitation, low rates of major vascular complication, mortality and stroke within 30 days with no difference between the 3 valve sizes.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02162069","term_id":"NCT02162069"}}NCT02162069     

Multiple roles of NF1 in the melanocyte lineage

NF1 is a tumour suppressor gene, germline mutations of which lead to neurofibromatosis type 1 syndrome. Patients develop benign tumours from several types of cells including neural crest‐derived cells. NF1 somatic mutations also occur in 15% of sporadic melanoma, a cancer originating from melanocytes. Evidence now suggests the involvement of NF1 mutations in melanoma resistance to targeted therapies. Although NF1 is ubiquitously expressed, genetic links between NF1 and genes involved in melanocyte biology have been described, implying the lineage‐specific mechanisms. In this review, we summarize and discuss the latest advances related to the roles of NF1 in melanocyte biology and in cutaneous melanoma.