Sodium deoxycholate causes nitric oxide mediated DNA damage in oesophageal cells

Patients with chronic gastro-oesophageal reflux disease experience the reflux of acid and bile into the distal oesophagus. The secondary bile salt sodium deoxycholate (NDC) is implicated in the induction of mucosal injury during reflux episodes. This study hypothesized that NDC damages DNA in oesophageal cells by an oxidative mechanism. In the oesophageal cell line HET1-A, increased production of nitric oxide (NO) was measured in NDC-treated cells. Protection from DNA strand breaks induced by NDC (10 µm) was observed in cells coincubated with the nitric oxide scavenger C-PTIO (p<0.012) or pre-incubated with the NO synthase inhibitor L-NAME (p<0.009) or the NFκB inhibitor, TPCK (p<0.036). Collectively these data implicate the involvement of NFκB and nitric oxide synthase in the DNA damage induced by NDC in oesophageal cells. In conclusion, NDC-driven NO production may play an important role in inducing DNA damage during episodes of gastro-oesophageal reflux and thereby contribute to reflux-related carcinogenesis.     

The puzzle of TRPV4 channelopathies

Hereditary channelopathies, that is, mutations in channel genes that alter channel function and are causal for the pathogenesis of the disease, have been described for several members of the transient receptor potential channel family. Mutations in the TRPV4 gene, encoding a polymodal Ca²⁺ permeable channel, are causative for several human diseases, which affect the skeletal system and the peripheral nervous system, with highly variable phenotypes. In this review, we describe the phenotypes of TRPV4 channelopathies and overlapping symptoms. Putative mechanisms to explain the puzzle, and how mutations in the same region of the channel cause different diseases, are discussed and experimental approaches to tackle this surprising problem are suggested.     

Biological and biochemical characterization of HIV‐1 Gag/dgp41 virus‐like particles expressed in Nicotiana benthamiana

The transmembrane HIV‐1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4+ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus‐like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant‐optimized HIV‐1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus‐based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV‐1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV‐1.     

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca²⁺ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).¹ Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca²⁺ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca²⁺-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 μM Ca²⁺. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca²⁺ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca²⁺ and Ca²⁺ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca²⁺ transient measurements, performed in these isolated myocytes.     

Understanding the Reduced Efficiencies of Organic Solar Cells Employing Fullerene Multiadducts as Acceptors

The use of fullerenes with two or more adducts as acceptors has been recently shown to enhance the performance of bulk‐heterojunction solar cells using poly(3‐hexylthiophene) (P3HT) as the donor. The enhancement is caused by a substantial increase in the open‐circuit voltage due to a rise in the fullerene lowest unoccupied molecular orbital (LUMO) level when going from monoadducts to multiadducts. While the increase in the open‐circuit voltage is obtained with many different polymers, most polymers other than P3HT show a substantially reduced photocurrent when blended with fullerene multiadducts like bis‐PCBM (bis adduct of Phenyl‐C₆₁‐butyric acid methyl ester) or the indene C₆₀ bis‐adduct ICBA. Here we investigate the reasons for this decrease in photocurrent. We find that it can be attributed partly to a loss in charge generation efficiency that may be related to the LUMO‐LUMO and HOMO‐HOMO (highest occupied molecular orbital) offsets at the donor‐acceptor heterojunction, and partly to reduced charge carrier collection efficiencies. We show that the P3HT exhibits efficient collection due to high hole and electron mobilities with mono‐ and multiadduct fullerenes. In contrast the less crystalline polymer Poly[[9‐(1‐octylnonyl)‐9H‐carbazole‐2,7‐diyl]‐2,5‐thiophenediyl‐2,1,3‐benzothiadiazole‐4,7‐diyl‐2,5‐thiophenediyl (PCDTBT) shows inefficient charge carrier collection, assigned to low hole mobility in the polymer and low electron mobility when blended with multiadduct fullerenes.     

Control of cardiac contraction by sodium: Promises,reckonings, and new beginnings

Several generations of cardiac physiologists have verified that basal cardiac contractility depends strongly on the transsarcolemmal Na gradient, and the underlying molecular mechanisms that link cardiac excitation-contraction coupling (ECC) to the Na gradient have been elucidated in good detail for more than 30 years. In brief, small increases of cytoplasmic Na push cardiac (NCX1) Na/Ca exchangers to increase contractility by increasing the myocyte Ca load. Accordingly, basal cardiac contractility is expected to be physiologically regulated by pathways that modify the cardiac Na gradient and the function of Na transporters. Assuming that this expectation is correct, it remains to be elucidated how in detail signaling pathways affecting the cardiac Na gradient are controlled in response to changing cardiac output requirements. Some puzzle pieces that may facilitate progress are outlined in this short review. Key open issues include (1) whether the concept of local Na gradients is viable, (2) how in detail Na channels, Na transporters and Na/K pumps are regulated by lipids and metabolic processes, (3) the physiological roles of Na/K pump inactivation, and (4) the possibility that key diffusible signaling molecules remain to be discovered.     

Sweat rate and sweat composition during heat acclimation

The purpose of this study was to determine local sweat rate (LSR) and sweat composition during heat acclimation (HA). For ten consecutive days of HA, eight participants cycled in 33 °C and 65% relative humidity at an intensity such that a rectal temperature of 38.5 °C was reached within ~40 min, followed by a 60-min clamp of this rectal temperature (i.e., controlled hyperthermia). Four participants extended HA by a 28-day decay period and five consecutive days of heat re-acclimation (HRA) using controlled hyperthermia. Sweat from the upper arm and upper back was collected three times during each heat exposure session. LSR and sweat sodium, chloride, lactate, and potassium concentrations were determined. Relative to HA day 1, LSR was increased at the final day of HA (day 10) (arm: +58%, P < 0.001; back: +36%, P < 0.05). Concentrations of sodium, chloride, and lactate significantly (P < 0.05) decreased to ~60% at HA day 10 compared to day 1 on the arm and back. Potassium concentration did not significantly differ on HA day 10 compared to day 1 (arm: +11%, P > 0.05; back: +8%, P > 0.05). The induction patterns of the sudomotor adaptations were different. Whilst LSR increased from HA day 8 on the arm and from HA day 7 on the back, sodium and chloride conservation already occurred from HA day 3 on both skin sites. Lastly, the sweat lactate reduction occurred from HA day 6 on the arm and back. Initial evidence is provided that adaptations were partly conserved after decay (28 days) and that a 5-day HRA may be sufficient to restore HA adaptations. In conclusion, ten days of exercise-induced HA using controlled hyperthermia led to increases in LSR and concomitant reductions of sweat sodium, chloride, and lactate concentrations, whilst potassium concentrations remained relatively constant.