Study on the effects of chloride depletion on photosystem II using different chloride depletion methods

Chloride is an indispensable factor for the functioning of oxygen evolving complex (OEC) and has protective and activating effects on photosystem II. In this study we have investigated mainly by EPR, the properties of chloride-sufficient, chloride-deficient and chloride-depleted thylakoid membranes and photosystem II enriched membranes from spinach. The results on the effects of different chloride depletion methods on the structural and functional aspects of photosystem II showed that chloride-depletion by treating PS II membranes with high pH is a relatively harsh way causing a significant and irreparable damage to the PS II donor side. Damage to the acceptor side of PS II was recovered almost fully in chloride-deficient as well as chloride-depleted PS II membranes.     

Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C₁ cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C₁ was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10) (m) increased furosemideinsensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanideinsensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloridedepleted cells. When cells with normal chloride content were depolarized (135 mm medium potassium + 10 m valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl^–Br^–>NO ₃ ^– >I^–. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.     

Effect of potassium depletion of Hep 2 cells on intracellular pH and on chloride uptake by anion antiport

The effect of K+ depletion of Hep 2 cells on ion fluxes, internal pH, cell volume, and membrane potential was studied. The cells were depleted of K+ by incubation in K+-free buffer with or without a preceding exposure to hypotonic medium. Efflux of K+ in cells not exposed to hypotonic medium was inhibited by furosemide or by incubation in Na+-free medium, indicating that in this case at least part of the K+ efflux occurs by Na+/K+/Cl- cotransport. After exposure to hypotonic medium, K+ efflux was not inhibited by furosemide, whereas it was partly inhibited by 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS). Exposure to hypotonic medium induced acidification of the cytosol, apparently because of efflux of protons from intracellular acidic vesicles. When isotonicity was restored, a rebound alkalinization of the cytosol was induced, because of activation of the Na+/H+ antiporter. While hypotonic shock and a subsequent incubation in K+-free buffer rapidly depolarized the cells, depolarization occurred much more slowly when the K+ depletion was carried out by incubation in K+-free buffer alone. The cell volume was reduced in both cases. K+ depletion by either method strongly reduced the ability of the cells to accumulate 36Cl- by anion antiport, and K+-depleted cells were unable to increase the rate of 36Cl- uptake in response to alkalinization of the cytosol.     

GABA reverse transport by the neuronal cotransporter GAT1: influence of internal chloride depletion

The role of intracellular ions on the reverse GABA transport by the neuronal transporter GAT1 was studied using voltage-clamp and [(3)H]GABA efflux determinations in Xenopus oocytes transfected with heterologous mRNA. Reverse transport was induced by intracellular GABA injections and measured in terms of the net outward current generated by the transporter. Changes in various intracellular ionic conditions affected the reverse current: higher concentrations of Na(+) enhanced the ratio of outward over inward transport current, while a considerable decrease of the outward current and a parallel reduction of the transporter-mediated GABA efflux were observed after treatments causing a diminution of the intracellular Cl(-) concentration. Particularly interesting was the impairment of the reverse transport observed after depletion of internal Cl(-) generated by the activity of a coexpressed K(+)-Cl(-) exporter KCC2. This finding suggests that reverse GABA transport may be physiologically regulated during early neuronal development, similarly to the functional alterations seen in GABA receptors caused by KCC2 activity.     

Slow potential changes in mammalian muscle fibers during prolonged hyperpolarization: Transport number effects and chloride depletion

Summary Mammalian skeletal muscle fibers exhibit large slow changes in membrane potential when hyperpolarized in standard chloride solutions. These large slow potential changes are radically reduced in low chloride solutions, where the faster and smaller potential change (creep), usually observed in amphibian fibers, becomes apparent. The slow potential change during a hyperpolarizing current pulse leads to an increase in apparent resistance of up to nine times the instantaneous value and takes minutes to reach a steady value. It then takes a similar time to decay very slowly back to the resting membrane potential after the current pulse. The halftime for the slow potential change was found to be inversely proportional to the current magnitude. From measurements of immediate postpulse membrane potentials, assuming constant ionic permeabilities, the internal chloride concentration was calculated to decrease exponentially towards a steady value (e.g., for one fiber from 12.3 to 6.6mm after a 330-sec pulse). The time course and magnitude of the concentration change were predicted from chloride transport number differences, and the known and measured properties of the fibers, and were found to agree very well with the values obtained from experimental measurements. In addition, the shapes of theV ₂-V ₁ responses, measured in the three-electrode current clamp set-up with either potassium chloride or potassium citrate current electrodes, were as predicted by transport number chloride depletion effects and were at variance with the predictions of a permeability change mechanism.     

The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion

The renal Na(+):Cl(-) cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl(-) influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl(-) efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.     

Heat inactivation of oxygen evolution in Photosystem II particles and its acceleration by chloride depletion and exogenous manganese

Heat inactivation of oxygen evolution by isolated Photosystem II particles was accelerated by Cl⁻ depletion and exogenous Mn²⁺. Weak red light also accelerated heat inactivation. Heat treatment released the 33, 24 and 18 kDa proteins and Mn from the Photosystem II particles. The protein release was stimulated by Cl⁻ depletion and exogenous Mn²⁺, and the Mn release was also stimulated by Cl⁻ depletion. A 50% loss of Mn corresponded to full inactivation of oxygen evolution, whereas no direct correlation seemed to exist between the loss of any one protein and inactivation of oxygen evolution. Removal of the 24 and 18 kDa proteins from photosystem II particles only slightly decreased the heat stability of oxygen evolution.     

Effect of sodium and chloride depletion on urinary prostaglandin F2α excretion in potassium loaded rats

Previous studies have shown that the urinary excretion of prostaglandin (PG) F2 alpha is stimulated by potassium (K) loading. Because changes of sodium chloride (NaCl) intake also affect renal PG production, in this study we investigated the interaction between the effect of K and that of concomitant reduction of Na and Cl intake. The urinary excretion of PGF2 alpha and PGE2 was measured in 12 groups of female rats on normal, high or low K intake. Na and Cl intake were adjusted so that rats had normal intake (controls, C), were selectively Cl depleted (CD), selectively Na depleted (ND) or Na and Cl depleted (NCD). In rats with normal K intake, urinary PGF2 alpha was not modified by changes of Na or Cl intake, whereas PGE2 was increased in by Cl depletion (in both NCD or CD groups). Potassium chloride loading increased urinary PGF2 alpha and selective Na depletion (ND group) induced a further increase. On the other hand, PGF2 alpha was not stimulated when K load was associated with Cl depletion. Urine PGF2 alpha was directly correlated with plasma aldosterone and urinary kallikrein. Urinary PGE2 did not change with K-loading. The results suggest that PGF2 alpha participates in the renal adaptation to KCl-loading but not when K is accompanied by non-Cl anions.     

Stratospheric ozone depletion

Solar ultraviolet radiation creates an ozone layer in the atmosphere which in turn completely absorbs the most energetic fraction of this radiation. This process both warms the air, creating the stratosphere between 15 and 50 km altitude, and protects the biological activities at the Earth's surface from this damaging radiation. In the last half-century, the chemical mechanisms operating within the ozone layer have been shown to include very efficient catalytic chain reactions involving the chemical species HO, HO2, NO, NO2, Cl and ClO. The NOX and ClOX chains involve the emission at Earth's surface of stable molecules in very low concentration (N2O, CCl2F2, CCl3F, etc.) which wander in the atmosphere for as long as a century before absorbing ultraviolet radiation and decomposing to create NO and Cl in the middle of the stratospheric ozone layer. The growing emissions of synthetic chlorofluorocarbon molecules cause a significant diminution in the ozone content of the stratosphere, with the result that more solar ultraviolet-B radiation (290-320 nm wavelength) reaches the surface. This ozone loss occurs in the temperate zone latitudes in all seasons, and especially drastically since the early 1980s in the south polar springtime-the 'Antarctic ozone hole'. The chemical reactions causing this ozone depletion are primarily based on atomic Cl and ClO, the product of its reaction with ozone. The further manufacture of chlorofluorocarbons has been banned by the 1992 revisions of the 1987 Montreal Protocol of the United Nations. Atmospheric measurements have confirmed that the Protocol has been very successful in reducing further emissions of these molecules. Recovery of the stratosphere to the ozone conditions of the 1950s will occur slowly over the rest of the twenty-first century because of the long lifetime of the precursor molecules.     

Effects of soil and wood depletion on biodiversity

Human depletion of soil and wood resources is dramatically altering the biodiversity of both terrestrial and aquatic ecosystems. This paper provides an overview of the numerous linkages between the depletion of soil and wood resources and the loss of biodiversity. While some of these linkages are well documented, others remain speculative or unexplored. In order to understand the full ramifications of resource depletion on biodiversity, additional research is required.     

Selenium depletion in patients on home parenteral nutrition

Severe selenium (Se) depletion was found in nine patients receiving long-term home parenteral nutrition because of short bowel syndrome. Plasma Se ranged from 0–0.51 (median 0.21 μmol/L) and erythrocyte Se ranged from 0.7–2.6 (median 1.8 μmol/gHgb), which was significantly lower than in the controls. Glutathione peroxidase (GSHPx) in plasma and erythrocytes was also decreased. After bolus injections with 200 μg Se/d in the form of sodium selenite for 4 mo, followed by 100 μg/d for 8 mo, plasma Se increased to values slightly but significantly higher than in the controls. Erythrocyte Se reached normal levels in most of the patients after 4 mo substitution, but it remained lower than in the controls. Following Se supplementation, plasma and erythrocyte GSHPx did not differ between patients and controls. These data suggest that all patients receiving long-term parenteral nutrition because of short bowel syndrome should receive at least 100 μg sodium selenite/d when given as bolus injections to avoid Se depletion.     

The effect of Golgi depletion on exocytic transport

Here we evaluate the idea that the Golgi is in dynamic equilibrium with the endoplasmic reticulum (ER). In cytoplasts that lack the Golgi apparatus, no regrowth of the Golgi is observed, nor is any transport from the ER to the cell surface detected. However, introduction of the smallest measurable amount of Golgi (equivalent to a few per cent per cell) yields significant exocytic transport. Our results indicate that the steady-state levels of Golgi in the ER are far smaller than the 30% that has been postulated, and that the Golgi may be an independent organelle and not simply an extension of the ER.     

Impact of benzalkonium chloride,benzethonium chloride and chloroxylenol on bacterial antimicrobial resistance

This review examined 3655 articles on benzalkonium chloride (BKC), benzethonium chloride (BZT) and chloroxylenol (CHO) aiming to understand their impact on antimicrobial resistance. Following the application of inclusion/exclusion criteria, only 230 articles were retained for analysis; 212 concerned BKC, with only 18 for CHO and BZT. Seventy-eight percent of studies used MIC to measure BKC efficacy. Very few studies defined the term ‘resistance’ and 85% of studies defined ‘resistance’ as <10-fold increase (40% as low as 2-fold) in MIC. Only a few in vitro studies reported on formulated products and when they did, products performed better. In vitro studies looking at the impact of BKC exposure on bacterial resistance used either a stepwise training protocol or exposure to constant BKC concentrations. In these, BKC exposure resulted in elevated MIC or/and MBC, often associated with efflux, and at time, a change in antibiotic susceptibility profile. The clinical relevance of these findings was, however, neither reported nor addressed. Of note, several studies reported that bacterial strains with an elevated MIC or MBC remained susceptible to the in-use BKC concentration. BKC exposure was shown to reduce bacterial diversity in complex microbial microcosms, although the clinical significance of such a change has not been established. The impact of BKC exposure on the dissemination of resistant genes (notably efflux) remains speculative, although it manifests that clinical, veterinary and food isolates with elevated BKC MIC carried multiple efflux pump genes. The correlation between BKC usage and gene carriage, maintenance and dissemination has also not been established. The lack of clinical interpretation and significance in these studies does not allow to establish with certainty the role of BKC on AMR in practice. The limited literature and BZT and CHO do not allow to conclude that these will impact negatively on emerging bacterial resistance in practice.     

Cord blood glutathione depletion in preterm infants: correlation with maternal cysteine depletion

Background

Depletion of blood glutathione (GSH), a key antioxidant, is known to occur in preterm infants.

Objective

Our aim was to determine: 1) whether GSH depletion is present at the time of birth; and 2) whether it is associated with insufficient availability of cysteine (cys), the limiting GSH precursor, or a decreased capacity to synthesize GSH.

Methodology

Sixteen mothers delivering very low birth weight infants (VLBW), and 16 mothers delivering healthy, full term neonates were enrolled. Immediately after birth, erythrocytes from umbilical vein, umbilical artery, and maternal blood were obtained to assess GSH [GSH] and cysteine [cys] concentrations, and the GSH synthesis rate was determined from the incorporation of labeled cysteine into GSH in isolated erythrocytes ex vivo, measured using gas chromatography mass spectrometry.

Principal Findings

Compared with mothers delivering at full term, mothers delivering prematurely had markedly lower erythrocyte [GSH] and [cys] and these were significantly depressed in VLBW infants, compared with term neonates. A strong correlation was found between maternal and fetal GSH and cysteine levels. The capacity to synthesize GSH was as high in VLBW as in term infants.

Conclusion

The current data demonstrate that: 1) GSH depletion is present at the time of birth in VLBW infants; 2) As VLBW neonates possess a fully active capacity to synthesize glutathione, the depletion may arise from inadequate cysteine availability, potentially due to maternal depletion. Further studies would be needed to determine whether maternal-fetal cysteine transfer is decreased in preterm infants, and, if so, whether cysteine supplementation of mothers at risk of delivering prematurely would strengthen antioxidant defense in preterm neonates.