Signaling pathway involved in the inhibitory effect of FTY720P on the Na<Superscript>+</Superscript>/K<Superscript>+</Superscript> ATPase in HepG2 cells

The Na⁺/K⁺ ATPase modulates the activity of many transporters in the liver, and maintains the ionic constancy of the intracellular milieu, preserving thus normal functioning of hepatocytes. Previous work showed that FTY720P, a sphingosine one phosphate receptor agonist used in the treatment of multiple sclerosis, exerts in HepG2 cells, an inhibitory effect on the activity of the ATPase, mediated via PGE2. This study is an attempt to identify the signaling molecules involved downstream of the prostaglandin. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme. The effect of FTY720P and PGE2 disappeared completely in presence of PF-04418948, a blocker of EP2 receptors, RpcAMP, an inhibitor of PKA, PD98059, an inhibitor of ERK, as well as in presence of PTIO, a nitric oxide synthase inhibitor, but was mimicked by butaprost, an EP2 agonist, dbcAMP, a cell permeable cAMP analogue, and SNAP1,a nitric oxide generator. PGE2 and dbcAMP increased the expression of phosphorylated ERK but not total ERK. This increase did not appear however in presence of PTIO, indicating that PKA is upstream of NO. It was concluded that FTY 720P induces PGE2 release which activates NOS leading to NO production and ERK activation. ERK then inhibits directly or indirectly the Na⁺/K⁺ ATPase.     

Activation of sarcolemma and nuclear membranes ET-1 receptors regulates transcellular calcium levels in heart and vascular smooth muscle cells

The use of an ET-1 fluorescent probe in human heart and vascular smooth muscle cells showed that ET-1 receptors are present at both the sarcolemma and nuclear envelope membranes. The use of immunofluorescence studies showed that the ETA receptor was mainly present at the sarcolemma and cytosolic levels. However, the ETB receptor was present at the sarcolemma and the cytosol, as well as the nuclear envelope membranes and the nucleoplasm. In addition, ET-1 immunoreactivity was seen in the cytosol and the nucleus. Using Ca2+ fluorescent probes such as Fluo-3, Indo 1, and yellow cameleon, as well as confocal microscopy three-dimensional image measurement technique, stimulation of ET-1 receptors at the sarcolemma membranes induced an increase of cytosolic and nuclear free Ca2+ levels. This effect of extracellular ET-1 was blocked by removal of extracellular calcium. Direct stimulation of ET-1 receptors at the nuclear envelope membranes also induced an increase of intranuclear free Ca2+ level. Our results suggest that the stimulation of sarcolemmal Ca2+ influx by ET-1 seems to be due to the activation of ETA and ETB receptors. However, the increase of nucleoplasmic Ca2+ levels by cytosolic ET-1 seems to be mediated via the activation of ETB receptors. Activation of nuclear membranes ETB receptors seems to prevent nuclear Ca2+ overload and may protect the cell from apoptosis.     

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.     

Sesquiterpene lactones from Sonchus macrocarpus

Eight sesquiterpene lactones were isolated from the roots of Sonchus macrocarpus. The eudesmanolides 15-hydroxy-4β, 15-dihydroreynosin and 15-hydroxy-4β, 15, 11β, 13-tetrahydroreynosin were isolated for the first time.     

Encapsulation of β-carotene in Nanoemulsion-Based Delivery Systems Formed by Spontaneous Emulsification: Influence of Lipid Composition on Stability and Bioaccessibility

Nanoemulsion-based delivery systems are finding increasing use in food, pharmaceutical, agrochemical, and personal care applications due to their ability to increase the stability and/or activity of lipophilic functional components. In this study, a low-energy homogenization method (spontaneous emulsification) was used to encapsulate β-carotene in nanoemulsions. The main objective was to optimize lipid phase composition to form stable nanoemulsions that would effectively enhance β-carotene bioavailability. Lipid phase composition was varied by mixing long chain triglycerides (LCT) with medium chain triglycerides (MCT) or flavor oil (orange oil). LCT was added to promote bioaccessibility, whereas MCT or orange oil was added to facilitate nanoemulsion formation. Our hypothesis was that an optimum level of LCT is required to form stable nanoemulsions with good bioaccessibility characteristics. Stable nanoemulsions could be formed at LCT-to-orange oil ratios of 1:1 (d ₃₂ = 109 nm) and at LCT-to-MCT ratios of 1:2 (d ₃₂ = 145 nm). Thus, higher LCT loading capacities and smaller droplet sizes could be obtained using orange oil. The influence of oil composition on the potential gastrointestinal fate of the nanoemulsions was studied using a simulated gastrointestinal tract (GIT) consisting of mouth, stomach, and small intestine phases. The transformation and bioaccessibility of β-carotene in the GIT was highly dependent on lipid phase composition. In particular, β-carotene bioaccessibility increased with increasing LCT level due to greater solubilization in mixed micelles. These results are useful for optimizing the design of nanoemulsion-based delivery systems for encapsulation and release of lipophilic nutraceuticals and pharmaceuticals.     

Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles,monomolecular films and UV-absorbing surface-coated substrate

Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10?ng of enzymes, against 100?ng to 10?μg with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate.     

One-Stage vs. Two-Stage Brachio-Basilic Arteriovenous Fistula for Dialysis Access: A Systematic Review and a Meta-Analysis

Introduction

A brachiobasilic arteriovenous fistula (BB-AVF) can provide access for haemodialysis in patients who are not eligible for a more superficial fistula. However, it is unclear whether one- or two-stage BB-AVF is the best option for patients.

Aim

To systematically assess the difference between both procedures in terms of access maturation, patency and postoperative complications.

Methods

Online search for randomised controlled trials (RCTs) and observational studies that compared the one-stage versus the two-stage technique for creating a BB-AVF.

Results

Eight studies were included (849 patients with 859 fistulas), 366 created using a one-stage technique, while 493 in a two-stage approach. There was no statistically significant difference between the two groups in the rate of successful maturation (Pooled risk ratio = 0.95 [0.82, 1.11], P = 0.53). Similarly, the incidence of postoperative haematoma (Pooled risk ratio = 0.73 [0.34, 1.58], P = 0.43), wound infection (Pooled risk ratio = 0.77 [0.35, 1.68], P = 0.51) and steal syndrome (Pooled risk ratio = 0.65 [0.27, 1.53], P = 0.32) were statistically comparable.

Conclusion

Although more studies seem to favour the two-stage BVT approach, evidence in the literature is not sufficient to draw a final conclusion as the difference between the one-stage and the two-stage approaches for creation of a BB-AVF is not statistically significant in terms of the overall maturation rate and postoperative complications. Patency rates (primary, assisted primary and secondary) were comparable in the majority of studies. Large randomised properly conducted trials with superior methodology and adequate sub-group analysis are needed before making a final recommendation.     

Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.