On the toxic effects of tetraethyl lead and its derivatives on the chrysophyte poterioochromonas malhamensis. IV. influence of lead antidotes and related agents

The unicellular alga Poterioochromonas malhamensis was exposed to 12.5 μM of inorganic or triethyl lead and simultaneously treated with lead antidotes and related agents at concentrations of 12.5, 31.25 and 62.5 μM. With increasing concentrations some of the antidotes alone slightly to severely inhibited algal growth (BAL¹, CaNa₂EDTA, EDTA, Na₂EDTA), whereas others (DPA, EGTA, DIZO) were non-toxic at the concentrations tested. EGTA and CaNa₂EDTA, at all concentrations tested, completely suppressed the growth inhibition caused by inorganic lead; Na₂EDTA and EDTA were protective at the lower or medium concentrations, but DIZO. DPA and BAL considerably enhanced lead toxicity with increasing concentrations. None of the tested agents was able to reduce the toxic effects of triethyl lead. All antidotes markedly increased inhibition of algal growth caused by triethyl lead and some were even lethal to the poisoned algae either at the highest (Na₂EDTA, EDTA, DPA) or at all concentrations used (DIZO, BAL). P. malhamensis proved to be a highly sensitive and valuable tests system and the results obtained exhibited striking parallels to medical and clinical experience in therapy of human poisoning with inorganic and organic lead compounds.     

Circadian Temperature and Activity Rhythms in Mice under Free-Running and Entrained Conditions; Assessment after Purification of the Temperature Rhythm

Six female mice were studied separately for six weeks, first in constant light (300 lx), and then on a 12 : 12 L : D schedule (light on 07:00–19:00–h). Food and water were available ad libitum. Abdominal temperature and spontaneous locomotor activity were measured every 10 min. In constant light, the animals free-ran with both temperature and activity records showing circadian rhythms that were significantly greater than 24 h; by contrast, in the LD schedule, the circadian rhythms had become entrained and showed a stable phase relation to this schedule. The direct masking effects upon raw temperatures caused by bursts of activity were clearly seen, and could be removed by a process of ‘purification’. A comparison of the activity profiles during the entrained and free-running phases showed that the imposed light-dark cycle resulted in decreased activity in the light, increased activity in the dark, and bursts of activity at the light-dark and dark-light transitions. Masking effects due to the activity profile were present in the raw temperature profile, and many could be removed by purification using the activity profile; however, there was evidence that other masking effects, independent of activity, were present also. The efficacy of thermoregulatory compensation, as assessed from the rise of core temperature produced by spontaneous locomotor activity, was, in comparison with the free-running condition, increased in the dark phase and decreased in the light phase; this would appear to be one way to limit the temperature rise that occurs in the active phase of the rest-activity cycle.     

Interaction between quaternary ammonium ions in the pore of potassium channels. Evidence against an electrostatic repulsion mechanism

We have examined the interaction between internal and external ions in the pore of potassium channels. We found that external tetraethylammonium was able to antagonize block of Shaker channels by internal TEA when the external and internal solutions contained K(+) ions. This antagonism was absent in solutions with Rb(+) as the only permeant ion. An externally applied trivalent TEA analogue, gallamine, was less effective than the monovalent TEA in inhibiting block by internal TEA. In addition, block by external TEA was little affected by changes in the concentration of internal K(+) ions, but was increased by the presence of internal Na(+) ions in the pore. These results demonstrate that external and internal TEA ions, likely located at opposite ends of the pore selectivity filter, do not experience a mutual electrostatic repulsion. We found that these results can be simulated by a simple 4-barrier-3-site permeation model in which ions compete for available binding sites without long-range electrostatic interactions.     

Serologic and immunochromatographic detection of oxygenated polyenoic acids in Euglena gracilis var. bacillaris

The extent of evolutionary conservation of DNA complimentary to RNA stored in the mature oocyte of the sea urchin S. purpuratus has been assessed. To do this, such DNA was hybridized with total genomic DNA of S. purpuratus and S. franciscanus and the thermal stability of the resultant duplexes was measured by two methods. In the first method, the duplexes were bound to hydroxylapatite and thermally eluted; the difference in thermal stability between homologous and heterologous duplexes averaged 6.9 degrees C in duplicate determinations. In the second experiment, the same hybrids were thermally melted in 2.4M tetraethylammonium chloride, then assayed with S1 nuclease; the difference in thermal stability of homologous and heterologous duplexes was 4.8 degrees C. Either value is significantly lower than the divergence of total single-copy DNA among these species as measured by the same techniques. This demonstrates that DNA sequences complimentary to maternal RNA are conserved during evolution, and thus that a high fraction of them are likely to be physiologically functional.     

Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes

Ion-selective microelectrodes are a powerful tool in studying adaptive responses of plant cells and tissues to various abiotic stresses. However, application of this technique in Na⁺ flux measurements was limited due to poor selectivity for Na⁺ ions of commercially available Na⁺ cocktails. Often, these cocktails cannot discriminate between Na⁺ and other interfering ions such as K⁺ and Ca²⁺, leading to inaccurate measurements of Na⁺ concentration and, consequently, inaccurate Na⁺ flux calculations. To overcome this problem, three Na⁺-selective cocktail mixtures were prepared using tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene. These cocktail mixtures were compared with commercially available ETH 227-based Na⁺ cocktail for selectivity for Na⁺ ions over other ions (particularly K⁺ and Ca²⁺). Among the three calixarene-based Na⁺ cocktails tested, cocktail 2 [in % w/w: Na⁺ ionophore (4-tert-butylcalix[4]arene-tetra acetic acid tetraethyl ester) 3.5, the plasticizer (2-nitrophenyl octyl ether) 95.9 and lipophilic anion (potassium tetrakis (4-chlorophenyl) borate) 0.6] showed the best selectivity for Na⁺ ions over K⁺ and Ca²⁺ ions and was highly stable over time (up to 10 h). Na⁺ flux measurements under a wide range of NaCl concentrations (25-150 mM) using Na⁺ cocktail 2 established a clear dose-response relationship between severity of salt stress and magnitude of Na⁺ influx at the distal elongation and mature zones of Arabidopsis thaliana roots. Furthermore, Na⁺ cocktail 2 was compared with commercially available ETH 227-based Na⁺ cocktail by measuring Na⁺ fluxes at the two Arabidopsis root zones in response to 100 mM NaCl treatment. With calixarene-based Na⁺ cocktail 2, a large decreasing Na⁺ influx (0-15 min) followed by small Na⁺ influx (15-45 min) was measured, whereas with ETH-based Na⁺ cocktail Na⁺ influx was short-lived (1-3 min) and was followed by Na⁺ efflux (3-45 min) that might have been due to K⁺ and Ca²⁺ efflux measured together with Na⁺ influx. In conclusion, Na⁺-selective calixarene-based microelectrodes have excellent potential to be used in real-time Na⁺ flux measurements in plants.     

Leishmania amazonensis infection may affect the ability of the host macrophage to be activated by altering their outward potassium currents

Understanding the impact of intracellular pathogens on the behaviour of their host cells is key to designing new interventions. We are interested in how Leishmania alters the electrical functioning of the plasma membrane of the macrophage it infects. The specific question addressed here is whether Leishmania amazonensis infection alters the macrophage’s outward currents and what the consequences of such changes might be. Using the whole cell configuration of the patch clamp technique, we show that outward peak current density remains constant over the period studied but that time to peak and sensitivity to inhibitors vary during infection. Infected cells take 40% longer to activate and are more sensitive to the potassium channel inhibitor tetraethyl ammonium, compared to control cells, indicating increased potassium outward current activity. Activation of macrophages is associated with increases of nitric oxide production and membrane area, depolarization of the macrophage membrane, down regulation of inward potassium and up regulation of outward currents. After Leishmania infection, macrophage activation is characterised by a reduction of nitric oxide production and of outward current density. We therefore suggest that this reflects a weaker activation.     

In crystallo capture of a Michaelis complex and product-binding modes of a bacterial phosphotriesterase

The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The μ-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that μ-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favour attack from a solvent molecule terminally coordinated to the α-metal ion. The bridging of both metal ions by the product, without disruption of the μ-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the β-metal ion, displacing the μ-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a μ-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK_a for the nucleophile, consistent with a general base function during catalysis.     

Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination

In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H⁺] by the catalytic reaction, measured by sol–gel entrapped indicators. The assay response at 562?nm was linear in the range pH?=?4–7. Linear calibration curves were obtained for α-endosulfan in the range of 0–30?μΜ. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N?=?28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps.     

Conformational properties of intrinsically disordered proteins bound to the surface of silica nanoparticles

Background

Protein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known.

Osmoregulation of leaf motor cells

"Osmotic Motors"--the best-documented explanation for plant leaf movements--frequently reside in specialized motor leaf organs, pulvini. The movements result from dissimilar volume and turgor changes in two oppositely positioned parts of the pulvinus. This Osmotic Motor is powered by a plasma membrane proton ATPase, which drives KCl fluxes and, consequently, water, across the pulvinus into swelling cells and out of shrinking cells. Light signals and signals from the endogenous biological clock converge on the channels through which these fluxes occur. These channels and their regulatory pathways in the pulvinus are the topic of this review.