Elevated ΔpH restores rapidly reversible photoprotective energy dissipation in <Emphasis Type="Italic">Arabidopsis</Emphasis> chloroplasts deficient in lutein and xanthophyll cycle activity

The xanthophylls of the light-harvesting complexes of photosystem II (LHCII), zeaxanthin, and lutein are thought to be essential for non-photochemical quenching (NPQ). NPQ is a process of photoprotective energy dissipation in photosystem II (PSII). The major rapidly reversible component of NPQ, qE, is activated by the transmembrane proton gradient, and involves the quenching of antenna chlorophyll excited states by the xanthophylls lutein and zeaxanthin. Using diaminodurene (DAD), a mediator of cyclic electron flow around photosystem I, to enhance ΔpH we demonstrate that qE can still be formed in the absence of lutein and light-induced formation of zeaxanthin in chloroplasts derived from the normally qE-deficient lut2npq1 mutant of Arabidopsis. The qE induced by high ΔpH in lut2npq1 chloroplasts quenched the level of fluorescence when all PSII reaction centers were in the open state (F _o state), protected PSII reaction centers from photoinhibition, was sensitive to the uncoupler nigericin, and was accompanied by absorption changes in the 410–565 nm region. Titrations show the ΔpH threshold for activation of qE in lut2npq1 chloroplasts lies outside the normal physiological range and is highly cooperative. Comparison of quenching in isolated trimeric (LHCII) and monomeric (CP26) light-harvesting complexes from lut2npq1 plants revealed a similarly shifted pH dependency compared with wild-type LHCII. The implications for the roles of lutein and zeaxanthin as direct quenchers of excitation energy are discussed. Furthermore, we argue that the control over the proton-antenna association constant, pK, occurs via influence of xanthophyll structure on the interconnected phenomena of light-harvesting antenna reorganization/aggregation and hydrophobicity.     

The effects of acute treatment with Δ9-THC on exploratory behaviour and memory in the rat

The aim of the present study was to evaluate the effects of delta9-tetrahydrocannabinol (delta9-THC) on exploratory behaviour and memory, independent of its locomotor suppressive effects. Dopamine (DA) and noradrenaline (NA) contents were determined in the areas of the brain directly related to such behaviours (hippocampus, striatum and amygdala). An acute dose of delta9-THC led to a decrease in exploratory parameters and motor activity during the holeboard test. The radial arm maze was used to evaluate the effects of this cannabinoid substance on memory. Animals treated with delta9-THC committed more errors in the maze test compared to control, particularly when the retention process was put to test. Furthermore, treatment with delta9-THC led to reduced NA contents in the hippocampus and increased DA contents in the amygdala, without changes in the striatum.     

Steady-state measurements of ΔpH and Δψ in Rhodospirillum rubrum chromatophores by two different methods. Comparison with phosphorylation potential

The pH gradient, ΔpH, and the membrane potential, Δψ, formed during light-induced electron transport in Rhodospirillum rubrum chromatophores were measured by two independent methods: (a) using specific electrodes to monitor light-dependent uptake of NH₄Cl and SCN^? at chromatophore concentrations of about 0.1 mg bacteriochlorophyll/ml and (b) using 9-aminoacridine and 8-anilinonaphthalenesulfonic acid as fluorescent probes for ΔpH and Δψ, respectively, at chromatophore concentrations of about 0.01 mg bacteriochlorophyll/ml. The light intensity was measured and set at a level which saturated the highest bacteriochlorophyll concentration used. The steady-state values obtained with each method under phosphorylating conditions were compared with the phosphorylation potential maintained by the chromatophores under identical conditions. The results indicate that under all conditions employed the ratio $\text{H}{}^{\mathrm{+}}\text{ATP}$ is greater than 2, and varies between 2.4 and 3.4 depending on the method used for estimation of the electrochemical proton gradient.     

Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes,pH and ionic strength on drug–receptor interactions

Nicotinic acetylcholine receptor (nAChR) α3-subunits, β4-subunits, α3/β4-subunit combination and α4/β2-subunit combination were immobilized on chromatographic stationary phases and the binding affinities of the different nAChR subtypes were chromatographically evaluated. The observed relative binding affinities of epibatidine were α4/β2>α3/β4 and epibatidine did not bind at α3-subunits and β4-subunits. No significant difference in binding affinities was observed on the α4/β2 nAChRs immobilized in immobilized artificial membrane (IAM) particles and those sterically immobilized on Superdex 200 beads. The effects of mobile phase pH and ionic strength on the binding affinities of the α3/β4 nAChRs support were also investigated. The results are consistent with the proposed ligand–nAChR binding model in which a cationic center exists at the binding site.     

Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.