Exposure to AC and DC magnetic fields induces changes in 5-HT1B receptor binding parameters in rat brain membranes

The binding properties of the G-protein coupled receptor (GPCR) serotonin 5-HT1B receptor were studied under exposure to AC (50 and 400 Hz) and DC magnetic fields (MF) in rat brain membranes. This was an attempt at replicating the positive findings of Massot et al. In saturation experiments using [3H]5-HT, 1-h exposures at 1.1 mT(rms) 50 Hz caused statistically significant increases in both the K(D) and B(max) binding parameters, from 1.74 +/- 0.3 to 4.51 +/- 0.86 nM and from 1428 +/- 205 to 2137 +/- 399 CPM, respectively, in good agreement with previous results. Exposure of the membranes at 400 Hz 0.675 mT(rms) did not elicit a larger increase in K(D) in spite of a much larger induced current density. DC fields (1.1 and 11 mT) had a lesser effect compared to AC fields at low values of K(Dsham), but decreased the affinity at higher values of K(Dsham). Modeling of the receptor-ligand-G protein interactions using the extended ternary complex model yielded good fits for all our data and that of Massot et al., showing that the AC field may act by decreasing the ability of the G-protein to alter the ligand-receptor affinity. The hypothesis is that the bipolar nature of the AC field explains the different nature of the effects observed with AC and DC exposures. These findings constitute one of the few documented pieces of evidence for cell-free effects of DC and extremely low frequency (ELF) AC MFs in the mT range.     

New evidence about the relationship between water channel activity and calcium in salinity-stressed pepper plants

This study, of how Ca2+ availability (intracellular, extracellular or linked to the membrane) influences the functionality of aquaporins of pepper (Capsicum annuum L.) plants grown under salinity stress, was carried out in plants treated with NaCl (50 mM), CaCl2 (10 mM), and CaCl2 (10 mM) + NaCl (50 mM). For this, water transport through the plasma membrane of isolated protoplasts, and the involvement of aquaporins and calcium (extracellular, intracellular and linked to the membrane) has been determined. After these treatments, it could be seen that the calcium concentration was reduced in the apoplast, in the cells and on the plasma membrane of roots of pepper plants grown under saline conditions; these concentrations were increased or restored when extra calcium was added to the nutrient solution. Protoplasts extracted from plants grown under Ca2+ starvation showed no aquaporin functionality. However, for the protoplasts to which calcium was added, an increase of aquaporin functionality of the plasma membrane was observed [osmotic water permeability (Pf) inhibition after Hg addition]. Interestingly, when verapamil (a Ca2+ channel blocker) was added, no functionality was observed, even when Ca2+ was added with verapamil. Therefore, calcium seems to be involved in plasma membrane aquaporin regulation via a chain of processes within the cell but not by alteration of the stability of the plasma membrane.     

Responses of wheat plants to nutrient deprivation may involve the regulation of water-channel function

The sap flow (Jv) and the osmotic hydraulic conductance (L₀) of detached, exuding root systems from wheat (Triticum aestivum L. cv. Chinese Spring) plants deprived of nitrogen for 5 d (— N) or of phosphorus for 7 d (—P), were measured and compared with controls receiving a complete nutrient supply. In the roots of — N and — P plants, Jv and L₀ decreased markedly, but between 4 and 24 h after resupplying N to — N plants (NRS plants) and P to — P plants (PRS plants), Jv and Lo recovered to values similar to those of control plants. Values of Jv and L₀ were always greater during the light period than during the dark, due to the diurnal variation of these parameters. Reducing transpiration in the light had no effect on Jv and L₀ of — N and — P plants. Sap flow and L₀ were also determined using individual axes from plants which had been grown with their roots divided between nutrient-deficient (- N or- P) solution and a complete nutrient solution. Differences were observed in Jv and L₀ between axes of the same plant, but stomatal conductance (Gs), which was also measured, was not affected in these split-root experiments. In control plants, Jv and L₀ declined sharply to values similar to those of roots from — N and — P plants after HgCl₂ treatment (50 M), but were restored by treating with 5 mM dithiothreitol. In plasma membranes from — N and — P roots, the amount of stigmasterol increased relative to sitosterol compared with control roots. The degree of unsaturation of bound fatty acids also increased, compared with controls, as a result of a decline in the relative amounts of 160 and 180 and an increase in 182. Plasma-membrane fluidity, estimated by steady-state fluorescence polarisation using 1,6-diphenyl hexatriene, showed that the plasma membranes from nutrient-deprived plants were less fluid than those from control plants, measured during both the light and dark periods and in split-root experiments. In NRS plants, the relative abundance of sitosterol increased, so that the stigmasterol/sitosterol ratio returned to a value similar to that of controls. However, in PRS plants, the difference in stigmasterol/sitosterol ratio was maintained, compared with controls. The degree of unsaturation of bound fatty acids, membrane fluidity and the hydraulic conductivity of root systems also recovered in NRS and PRS plants to values similar to those of control plant plasma membranes. The results obtained suggested that — N and — P treatment decreased L₀, by reducing either the activity or the abundance of Hg-sensitive water channels. Also, there may be an interaction between the increase in membrane lipid ordering and the decrease in L₀.Abbreviations DTT dithiothreitol - Gs stomatal conductance - Js solute flow into the sap - Jv sap flow - L₀ hydraulic conductance - - N nitrogen-deprived for 5 d - NRS nitrogen resupplied after 5 d deprivation - - P phosphorus-deprived for 7 d - PRS phosphorus resupplied after 7 d deprivation M.C. was funded by a grant from CSIC, Spain. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.     

Dynamics of a small re-introduced population of wild dogs over 25 years: Allee effects and the implications of sociality for endangered species’ recovery

We analysed 25 years (1980–2004) of demographic data on a small re-introduced population of endangered African wild dogs (Lycaon pictus) in Hluhluwe-iMfolozi Park (HiP), South Africa, to describe population and pack dynamics. As small populations of cooperative breeders may be particularly prone to Allee effects, this extensive data set was used to test the prediction that, if Allee effects occur, aspects of reproductive success, individual survival and population growth should increase with pack and population size. The results suggest that behavioural aspects of wild dogs rather than ecological factors (i.e. competitors, prey and rainfall) primarily have been limiting the HiP wild dog population, particularly a low probability of finding suitable mates upon dispersal at low pack number (i.e. a mate-finding Allee effect). Wild dogs in HiP were not subject to component Allee effects at the pack level, most likely due to low interspecific competition and high prey availability. This suggests that aspects of the environment can mediate the strength of Allee effects. There was also no demographic Allee effect in the HiP wild dog population, as the population growth rate was significantly negatively related to population size, despite no apparent ecological resource limitation. Such negative density dependence at low numbers indicates that behavioural studies of the causal mechanisms potentially generating Allee effects in small populations can provide a key to understanding their dynamics. This study demonstrates how aspects of a species’ social behaviour can influence the vulnerability of small populations to extinction and illustrates the profound implications of sociality for endangered species’ recovery. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Boric acid and salinity effects on maize roots. Response of aquaporins ZmPIP1 and ZmPIP2, and plasma membrane H+-ATPase, in relation to water and nutrient uptake

Under saline conditions, an optimal cell water balance, possibly mediated by aquaporins, is important to maintain the whole-plant water status. Furthermore, excessive accumulation of boric acid in the soil solution can be observed in saline soils. In this work, the interaction between salinity and excess boron with respect to the root hydraulic conductance (L₀), abundance of aquaporins (ZmPIP1 and ZmPIP2), ATPase activity and root sap nutrient content, in the highly boron- and salt-tolerant Zea mays L. cv. amylacea, was evaluated. A downregulation of root ZmPIP1 and ZmPIP2 aquaporin contents were observed in NaCl-treated plants in agreement with the L₀ measurements. However, in the H₃BO₃-treated plants differences in the ZmPIP1 and ZmPIP2 abundance were observed. The ATPase activity was related directly to the amount of ATPase protein and Na⁺ concentration in the roots, for which an increase in NaCl- and H₃BO₃+ NaCl-treated plants was observed with respect to untreated and H₃BO₃-treated plants. Although nutrient imbalance may result from the effect of salinity or H₃BO₃ alone, an ameliorative effect was observed when both treatments were applied together. In conclusion, our results suggest that under salt stress, the activity of specific membrane components can be influenced directly by boric acid, regulating the functions of certain aquaporin isoforms and ATPase as possible components of the salinity tolerance mechanism.     

Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites

Recently, several mutants of cytochrome P450 BM3 (CYP102A1) with high activity toward drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts in the production of reactive metabolites from the drugs clozapine, diclofenac and acetaminophen was investigated. We showed that the four CYP102A1 mutants used in this study formed the same metabolites as human and rat liver microsomes, with an activity up to 70-fold higher compared to human enzymes. Using these CYP102A1 mutants, three novels GSH adducts of diclofenac were discovered which were also formed in incubations with human liver microsomes. This work shows that CYP102A1 mutants are very useful tools for the generation of high levels of reference metabolites and reactive intermediates of drugs. Producing high levels of those reactive metabolites, that might play a role in adverse drug reactions (ADRs) in humans, will facilitate their isolation, structural elucidation, and could be very useful for the toxicological characterization of novel drugs and/or drug candidates.     

Inward rectifying potassium channels facilitate cell-to-cell communication in hamster retractor muscle feed arteries

This study examined whether inward rectifying K+ (KIR) channels facilitate cell-to-cell communication along skeletal muscle resistance arteries. With the use of feed arteries from the hamster retractor muscle, experiments examined whether KIR channels were functionally expressed and whether channel blockade attenuated the conduction of acetylcholine-induced vasodilation, an index of cell-to-cell communication. Consistent with KIR channel expression, this study observed the following: 1) a sustained Ba2+-sensitive, K+-induced dilation in preconstricted arteries; 2) a Ba2+-sensitive inwardly rectifying K+ current in arterial smooth muscle cells; and 3) KIR2.1 and KIR2.2 expression in the smooth muscle layer of these arteries. It was subsequently shown that the discrete application of acetylcholine elicits a vasodilation that conducts with limited decay along the feed artery wall. In the presence of 100 microM Ba2+, the local and conducted response to acetylcholine was attenuated, a finding consistent with a role for KIR in facilitating cell-to-cell communication. A computational model of vascular communication accurately predicted these observations. Control experiments revealed that in contrast to Ba2+, ATP-sensitive- and large-conductance Ca2+ activated-K+ channel inhibitors had no effect on the local or conducted vasodilatory response to acetylcholine. We conclude that smooth muscle KIR channels play a key role in facilitating cell-to-cell communication along skeletal muscle resistance arteries. We attribute this facilitation to the intrinsic property of negative slope conductance, a biophysical feature common to KIR2.1- and 2.2-containing channels, which enables them to increase their activity as a cell hyperpolarizes.     

Prnp knockdown in transgenic mice using RNA interference

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.     

Docking and hydropathic scoring of polysubstituted pyrrole compounds with antitubulin activity

Compounds that bind at the colchicine site of tubulin have drawn considerable attention with studies indicating that these agents suppress microtubule dynamics and inhibit tubulin polymerization. Data for 18 polysubstituted pyrrole compounds are reported, including antiproliferative activity against human MDA-MB-435 cells and calculated free energies of binding following docking the compounds into models of alphabeta-tubulin. These docking calculations coupled with HINT interaction analyses are able to represent the complex structures and the binding modes of inhibitors such that calculated and measured free energies of binding correlate with an r(2) of 0.76. Structural analysis of the binding pocket identifies important intermolecular contacts that mediate binding. As seen experimentally, the complex with JG-03-14 (3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester) is the most stable. These results illuminate the binding process and should be valuable in the design of new pyrrole-based colchicine site inhibitors as these compounds have very accessible syntheses.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.