Lumen pH Research Articles

PS II model-based simulations of single turnover flash-induced transients of fluorescence yield monitored within the time domain of 100 ns–10 s on dark-adapted Chlorella pyrenoidosa cells

The set up described in Steffen et al. (Biochemistry 40:173-180, 2001) was used to monitor in the time domain from 100 ns to 10 s single turnover flash-induced transients of the normalized fluorescence yield (SFITFY) on dark-adapted cells of the thermophilic algae Chlorella pyrenoidosa Chick. Perfect data fit was achieved within the framework of a previously proposed model for the PS II reaction pattern (Lebedeva et al., Biophysics 47:968-980, 2002; Belyaeva et al., Biophysics 51:860-872, 2006) after its modification by taking into account nonradiative decay processes including nonphotochemical quenching due to time-dependent populations of P680(+*) and (3)Car. On the basis of data reported in the literature, a consistent set of rate constants was obtained for electron transfer at the donor and acceptor sides of PS II, pH in lumen and stroma, the initial redox state of plastoquinone pool and the rate of plastoquinone oxidation. The evaluation of the rate constant values of dissipative processes due to quenching by carotenoid triplets in antennae and P680(+*)Q(A)(-*) recombination as well as the initial state populations after excitation with a single laser flash are close to that outlined in (Steffen et al., Biochemistry 44:3123-3133, 2005a). The simulations based on the model of the PS II reaction pattern provide information on the time courses of population probabilities of different PS II states. We analyzed the maximum (F(m)(STF)) and minimum (F(0)) of the normalized FL yield dependence on the rate of the recombination processes (radiative and dissipative nonradiative) and of P680(+*) reduction. The developed PS II model provides a basis for theoretical comparative analyses of time-dependent fluorescence signals, observed at different photosynthetic samples under various conditions (e.g. presence of herbicides, other stress conditions, excitation with actinic pulses of different intensity, and duration).

Guar Gum as Potential Film Coating Material for Colon-specific Delivery of Fluorouracil

The potential of guar gum as a film coating material for colon-specific delivery of 5-fluorouracil is evaluated in this study. The guar gum-based multi-unit pellet system is prepared by coating guar gum and pH-sensitive polymer Eudragit FS30D sequentially around drug-loaded non-pareil cores in a fluid-bed coater. The outer Eudragit FS coating protects the system against gastrointestinal environment and dissolves rapidly in distal small intestine, where a lumen pH of over 7 triggers the dissolution of the enteric polymer. The inner guar gum coating works as a time-controlled retardant and offers additional protection of the pellets until it is degraded by microbial enzymes at the proximal colon. In vitro results indicate that guar gum is a feasible coating material to achieve timed and enzyme-triggered fluorouracil release. Pharmacokinetic study in beagle dogs shows delayed absorption of about 5 h and limited absorption fraction as a result of guar gum and Eudragit FS coating.

Arabidopsis Genotypes with Differing Levels of psbS Expression Differ in Photosystem II Quantum Yield, Xanthophyll Cycle Pool Size, and Aboveground Growth

Thermal energy dissipation protects the photosynthetic apparatus against excess light and requires de‐epoxidized xanthophyll cycle carotenoids, a low thylakoid lumen pH, and PsbS, an integral membrane subunit of photosystem II. Here, we examine photoprotection, photosynthesis, and growth in Arabidopsis thaliana genotypes whose capacities for energy dissipation have been altered via deletion or transgenic overexpression of psbS. Plants were grown in a greenhouse, where they experienced low levels of incident radiation throughout most of the day, with an ∼40‐min midmorning period of exposure to high incident radiation each day. Midday ratio of variable to maximal fluorescence and photosystem II quantum yield increased slightly with increasing levels of psbS expression, while xanthophyll cycle pool size and conversion state were negatively associated with psbS expression. Notably, transgenic overexpression of psbS led to a 12% increase in rosette diameter, while mutants with no psbS expression exhibited 16% smaller rosette diameters and 18% fewer leaves than the wild type. Thus, the protective role of thermal energy dissipation manifested in plants grown in a light environment that included a period of high incident radiation, during which the various genotypes sustained differing levels of photosystem II photoinactivation, and periods of low incident radiation, when electron transport presumably limited the rate of photosynthesis and photosystem II photoinactivation influenced the overall rate of photosynthesis and hence growth.

Effect of nutrient mixture and black grapes on the pharmacokinetics of orally administered (‐)epigallocatechin‐3‐gallate from green tea extract: a human study

(-)Epigallocatechin-3-gallate (EGCG), a green tea component, has been attributed with anticarcinogenic and antioxidant activities. The extent and rate of absorption of EGCG by the small intestine depends on various factors such as molecular size, lipophilicity, solubility, pKa, gastric and intestinal transit time, lumen pH, membrane permeability and first pass metabolism. The bioavailability of EGCG can be increased by decreasing the presystemic elimination by stabilizing EGCG in the lumen, helping its transfer across the intestinal apical membrane and its accumulation and thus its availability by inhibiting phase I and II enzymes and phase III transporters. In a crossover study, five human volunteers were given a single oral dose of GTE (A), nutrient mixture (NM) containing GTE (B) and formulation B along with black grapes 250 g (C). Blood samples were drawn at 0, 2, 4, 6 and 8 h. The pharmacokinetic parameters were analysed by WinNonLin (Vs 5.0.1.) using a non-compartmental approach. Supplementation with nutrient mixture normally prescribed to cancer patients containing ascorbic acid, selenium, N-acetyl cysteine and other nutrients (formulation B) resulted in an increase of the systemic availability of EGCG by 14% and formulation C further increased it by 13%, thus leading to a total increase of 27%.

Peripheral membrane proteins mediate binding of vacuolar storage proteins to membranes of the secretory pathway of developing pea cotyledons.

In developing pea cotyledons, storage proteins are sorted via dense vesicles into the protein storage vacuole. Formation of these unique transport vesicles is characterized by aggregation of their cargo proteins. Protein sorting into dense vesicles is pH dependent. In order to gain insight into the molecular basis of storage protein sorting, a membrane binding assay was developed which allows for a detailed biochemical analysis of binding events. Employing this assay it was possible to show that storage proteins bind in a pH-dependent manner to the membranes of the secretory pathway with a pH optimum in the range of the lumenal pH of the Golgi cisternae. Through reconstitution experiments, it was possible to demonstrate further that this recruitment occurs via the interaction of peripheral rather than intrinsic membrane proteins. Results of co-immunoprecipitation experiments point to interactions between different storage proteins in the secretory system. These results are discussed in terms of the aggregation-mediated sorting of storage proteins into maturing dense vesicles.

Cationic pathway of pH regulation in larvae ofAnopheles gambiae

Anopheles gambiae larvae (Diptera: Culicidae) live in freshwater with low Na(+) concentrations yet they use Na(+) for alkalinization of the alimentary canal, for electrophoretic amino acid uptake and for nerve function. The metabolic pathway by which larvae accomplish these functions has anionic and cationic components that interact and allow the larva to conserve Na(+) while excreting H(+) and HCO(3)(-). The anionic pathway consists of a metabolic CO(2) diffusion process, carbonic anhydrase and Cl(-)/HCO(3)(-) exchangers; it provides weak HCO(3)(-) and weaker CO(3)(2-) anions to the lumen. The cationic pathway consists of H(+) V-ATPases and Na(+)/H(+) antiporters (NHAs), Na(+)/K(+) P-ATPases and Na(+)/H(+) exchangers (NHEs) along with several (Na(+) or K(+)):amino acid(+/-) symporters, a.k.a. nutrient amino acid transporters (NATs). This paper considers the cationic pathway, which provides the strong Na(+) or K(+) cations that alkalinize the lumen in anterior midgut then removes them and restores a lower pH in posterior midgut. A key member of the cationic pathway is a Na(+)/H(+) antiporter, which was cloned recently from Anopheles gambiae larvae, localized strategically in plasma membranes of the alimentary canal and named AgNHA1 based upon its phylogeny. A phylogenetic comparison of all cloned NHAs and NHEs revealed that AgNHA1 is the first metazoan NHA to be cloned and localized and that it is in the same clade as electrophoretic prokaryotic NHAs that are driven by the electrogenic H(+) F-ATPase. Like prokaryotic NHAs, AgNHA1 is thought to be electrophoretic and to be driven by the electrogenic H(+) V-ATPase. Both AgNHA1 and alkalophilic bacterial NHAs face highly alkaline environments; to alkalinize the larva mosquito midgut lumen, AgNHA1, like the bacterial NHAs, would have to move nH(+) inwardly and Na(+) outwardly. Perhaps the alkaline environment that led to the evolution of electrophoretic prokaryotic NHAs also led to the evolution of an electrophoretic AgNHA1 in mosquito larvae. In support of this hypothesis, antibodies to both AgNHA1 and H(+) V-ATPase label the same membranes in An. gambiae larvae. The localization of H(+) V-ATPase together with (Na(+) or K(+)):amino acid(+/-) symporter, AgNAT8, on the same apical membrane in posterior midgut cells constitutes the functional equivalent of an NHE that lowers the pH in the posterior midgut lumen. All NATs characterized to date are Na(+) or K(+) symporters so the deduction is likely to have wide application. The deduced colocalization of H(+) V-ATPase, AgNHA1 and AgNAT8, on this membrane forms a pathway for local cycling of H(+) and Na(+) in posterior midgut. The local H(+) cycle would prevent unchecked acidification of the lumen while the local Na(+) cycle would regulate pH and support Na(+):amino acid(+/-) symport. Meanwhile, a long-range Na(+) cycle first transfers Na(+) from the blood to gastric caeca and anterior midgut lumen where it initiates alkalinization and then returns Na(+) from the rectal lumen to the blood, where it prevents loss of Na(+) during H(+) and HCO(3)(-) excretion. The localization of H(+) V-ATPase and Na(+)/K(+)-ATPase in An. gambiae larvae parallels that reported for Aedes aegypti larvae. The deduced colocalization of the two ATPases along with NHA and NAT in the alimentary canal constitutes a cationic pathway for Na(+)-conserving midgut alkalinization and de-alkalinization which has never been reported before.

Photochemistry, remotely sensed physiological reflectance index and de-epoxidation state of the xanthophyll cycle in Quercus coccifera under intense drought

Quercus coccifera L. is a Mediterranean sclerophyllous shrub with a high capacity to resist intense drought stress. Therefore, it could be used in the study of physiological changes suffered by plants at very low water potentials. A remote sensing sensor was used to measure continuously the physiological reflectance index (PRI; defined as the changes in reflectance at 531 nm with respect to those at 570 nm; PRI = [(R531 - R570)/(R531 + R570)] at canopy level and under field conditions in an artificial carpet of seedlings of Q. coccifera during a drought cycle. Correlations between leaf level-measured chlorophyll fluorescence parameters as well as the de-epoxidation state of the xanthophyll cycle [(A + Z)/(V + A + Z)] and canopy level-measured PRI were reasonably good (R (2) = 0.57-0.63, P < 0.01), and quite interesting for water stress remote sensing purposes. The instrument's temporal resolution allowed us to follow the rapid response of PRI to changing photosynthetic active radiation, and to resolve, in response to cloud-induced changes in light intensity, a fast and a slow PRI component. We report the disappearance of the rapid one under conditions of intense drought in response to a sudden increase in light intensity. The underlying photoprotection mechanisms that Q. coccifera shows in response to intense drought stress periods seem to be related to the existence of a low intrathylakoid lumenal pH at the end of the drought cycle. Under intense drought, these mechanisms allow this species to avoid oxidative damage, which was evidenced by the maintenance of an unaltered photosynthetic pigment composition and constant photosystem II efficiency in the mornings. It is concluded that, contrary to early reports, PRI is a sensible, indirect, non-destructive water stress indicator, even in plants experiencing intense drought.

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with p K values in the range 4.7–7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1 H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1 H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1 H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH in established during illumination of chloroplasts with continuous light. For isolated bean ( Vicia faba) chloroplasts suspended in a medium with pH out = 7.8, we found that pH in ≈ 5.4–5.7 in the state of photosynthetic control, and pH in ≈ 5.7–6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8–2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H +/ATP is n ≥ 4–4.7.

A Report on Associations Among Gastric pH, Bleeding, Duodenogastric Reflux, and Outcomes After Trauma

The pathogenesis of multiple organ failure (MOF) in trauma patients may involve the gastrointestinal tract, but its exact origins remain elusive. In a prospective study, the gastric fluid of major torso trauma patients was examined for evidence of duodenogastric reflux and potential gastric injury, and was compared with patient outcomes regarding MOF. Patient samples were collected daily for 4 days by nasogastric tube and analyzed for pH, hemoglobin, and bile acid. Blood was collected for analysis of C-reactive protein (CRP). Outcomes were recorded for the presence or absence of MOF. The results showed that most patients exhibited alkaline gastric contents (pH >/=4.9) and elevated levels of hemoglobin immediately after the trauma. Although non-MOF patients demonstrated a decline of both mean gastric pH and bleeding by day 4, MOF patients maintained significant elevations in pH during this time period. Mean total bile acid levels were increased in all patients, signifying the presence of duodenogastric reflux. However, there were no clear differences in mean bile acid concentrations between MOF and non-MOF patients over time, although MOF patients tended to exhibit higher levels. All patients showed a progressive rise in serum CRP during the first 24 hours after trauma, which was maintained for 4 days. The initial rise in serum CRP in MOF patients was delayed compared with that in non-MOF patients. We conclude that duodenogastric reflux occurs in trauma patients in the first few days after trauma and may contribute to elevated gastric pH and bleeding. Further study is needed to verify whether monitoring the gastric juice of trauma patients during the first several days of hospitalization, for alkaline pH and excessive blood in the gastric lumen, could lead to better assessments of patient status.

Enhanced Formation of a HCO−3 Transport Metabolon in Exocrine Cells of Nhe1–/– Mice

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.

Trapping of the quenched conformation associated with non-photochemical quenching of chlorophyll fluorescence at low temperature.

The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25 degrees C and during rapid jumps of the leaf temperature. At 5 degrees C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25 degrees C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (delta A515) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201-4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE.

Influence of light-induced changes in stromal and lumenal pH on electron transport kinetics in chloroplasts: Mathematical modeling

A mathematical model of electron and proton transport in higher plant chloroplasts is built with allowance for light-induced pH changes in the stroma and the thylakoid lumen. The model considers the key steps of electron transport from photosystem II to NADP+, the terminal acceptor of PS I, ATP synthesis from ADP and inorganic phosphate coupled with transmembrane proton transport, consumption of NADPH and ATP in the Calvin cycle. Account of the influence of lumenal and stromal pH changes on the rate of electron transport at the plastoquinone segment of the chain and on the acceptor side of PS I allows description of the particular features of the complex kinetics of electron transport in intact chloroplasts. Calculations show that accelerated efflux of electrons at the acceptor part of PS I, resulting from activation of the Calvin cycle reaction upon light-induced stromal alkalization, may be one of the causes of the multiphasic kinetics of the redox conversions of the PS I primary donor P700, which is characteristic of dark-adapted cyanobacteria, algae, and intact higher plant chloroplasts.

The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced pmf

Endogenous probes of light-induced transthylakoid proton motive force ( pmf), membrane potential (Δ ψ) and ΔpH were used in vivo to assess in Arabidopsis the lumen pH responses of regulatory components of photosynthesis. The accumulation of zeaxanthin and protonation of PsbS were found to have similar p K a values, but quite distinct Hill coefficients, a feature allowing high antenna efficiency at low pmf and fine adjustment at higher pmf. The onset of “energy-dependent’ exciton quenching ( q E) occurred at higher lumen pH than slowing of plastoquinol oxidation at the cytochrome b 6 f complex, presumably to prevent buildup of reduced electron carriers that can lead to photodamage. Quantitative comparison of intrinsic probes with the electrochromic shift signal in situ allowed quantitative estimates of pmf and lumen pH. Within a degree of uncertainly of ∼ 0.5 pH units, the lumen pH was estimated to range from ∼ 7.5 (under weak light at ambient CO 2) to ∼ 5.7 (under 50 ppm CO 2 and saturating light), consistent with a ‘moderate pH’ model, allowing antenna regulation but preventing acid-induced photodamage. The apparent p K a values for accumulation of zeaxanthin and PsbS protonation were found to be ∼ 6.8, with Hill coefficients of about 4 and 1 respectively. The apparent shift between in vitro violaxanthin deepoxidase protonation and zeaxanthin accumulation in vivo is explained by steady-state competition between zeaxanthin formation and its subsequent epoxidation by zeaxanthin epoxidase. In contrast to tobacco, Arabidopsis showed substantial variations in the fraction of pmf (0.1–0.7) stored as Δ ψ, allowing a more sensitive qE response, possible as an adaptation to life at lower light levels.

In situ measurement of pH in the secreting canaliculus of the gastric parietal cell and adjacent structures

The gastric H(+)/K(+)-ATPase is located within an infolding (secretory canaliculus) of the apical plasma membrane of gastric parietal cells. Our aim was to measure the pH values in the cytosol and canaliculus of the acid-secreting parietal cell and the adjacent gland lumen in situ. We used ultrafine double-barreled tip-sealed microelectrodes at high acceleration rates for intracellular and canalicular measurements. Immunohistochemical staining of the parietal cells was used to identify the track of the electrode and to estimate the position of the electrode tip at the time of the last intracellular measurement. En route to the deepest regions of the mucosa, where the average gland lumen pH was approximately 3, and on advancing in steps of 2 mum, the electrode entered the cytosol of the parietal cells, where the pH value was 7.4. Advancing the electrode further resulted, in several instances, in a sharp decrease in pH to an average value of 1.7 +/- 0.2, which we interpreted as the measurement within the canaliculus. When the electrode was advanced even further, the pH reading returned to the cytosolic value. From the difference in pH between the secreting canaliculus and the adjacent gland lumen, we concluded that the released acid was immediately buffered. Thus, the only cellular structure directly exposed to the highly acidic canalicular content is the apical membrane forming the canaliculus in the parietal cell.

Turned On by a Low pH

Salmonellae , facultative intracellular pathogens that cause gastroenteritis and typhoid fever, respond to the normally inhospitable environment inside macrophage phagosomes--in which pathogens are exposed to antimicrobial peptides, reactive oxygen species, and acidic pH--by activating the transcription of virulence genes. This process, which depends on phagosomal acidification, involves the activation of the membrane-bound sensor kinase PhoQ, which leads to its autophosphorylation and the subsequent phosphorylation of the transcriptional activator PhoP (the PhoPQ system). Using a bacterial strain that expressed a reporter that was transcriptionally activated by PhoP, Prost et al . showed that, in the presence of 1 mM Mg 2+ (the estimated phagosomal concentration), transcription increased when the pH was decreased from 7.5 to 5.5. When PhoQ was reconstituted in vesicles so that the periplasmic sensor domain faced inward and the cytoplasmic kinase domain faced outward, PhoP phosphorylation increased when lumenal pH was decreased from 7.5 to 5.5. Subinhibitory concentrations of antimicrobial peptides have previously been shown to activate PhoQ through a mechanism that may involve displacement of divalent cations, and the effects of antimicrobial peptides and pH on PhoP phosphorylation and PhoPQ-dependent transcription were additive. Nuclear magnetic resonance (NMR) spectroscopy revealed that the conformation of the PhoQ sensor domain was affected by changes in pH and that this conformational change did not depend on loss of divalent cation binding. Thus, the authors propose that PhoQ acts as a pH sensor and that antimicrobial peptides and low pH work together in macrophage phagosomes to activate the PhoPQ system. L. R. Prost, M. E. Daley, V. Le Sage, M. W. Bader, H. Le Moual, R. E. Klevit, S. I. Miller, Activation of the bacterial sensor kinase PhoQ by acidic pH. Mol. Cell 26 , 165-174 (2007). [Online Journal]

The Transiently Generated Nonphotochemical Quenching of Excitation Energy in Arabidopsis Leaves Is Modulated by Zeaxanthin

Upon the transition of dark-adapted plants to low light, the energy-dependent quenching (qE) of excitation energy is only transiently induced due to the only transient generation of the transthylakoid pH gradient. We investigated the transient qE (qETR) in different Arabidopsis (Arabidopsis thaliana) mutants. In dark-adapted plants, qETR was absent in the npq4 mutant (deficient in the PsbS protein) and the pgr1 mutant (restricted in lumen acidification). In comparison with wild-type plants, qETR was reduced in the zeaxanthin (Zx)-deficient npq1 mutant and increased in the Zx-accumulating npq2 mutant. After preillumination of plants (to allow the synthesis of large amounts of Zx), the formation and relaxation of qETR was accelerated in all plants (except for npq4) in comparison with the respective dark-adapted plants. The extent of qETR, however, was unchanged in npq1 and npq4, decreased in npq2, but increased in wild-type and pgr1 plants. Even in presence of high levels of Zx, qETR in pgr1 mutants was still lower than that in wild-type plants. In the presence of the uncoupler nigericin, qETR was completely abolished in all genotypes. Thus, the transient qETR shows essentially the same characteristics as the steady-state qE; it is strictly dependent on the PsbS protein and a low lumen pH, but the extent of qETR is largely modulated by Zx. These results indicate that qETR does not represent a different quenching mechanism in comparison with the steady-state qE, but simply reflects the response of qE to the dynamics of the lumen pH during light activation of photosynthesis.

The transiently generated nonphotochemical quenching of excitation energy in Arabidopsis leaves is modulated by zeaxanthin.

Upon the transition of dark-adapted plants to low light, the energy-dependent quenching (qE) of excitation energy is only transiently induced due to the only transient generation of the transthylakoid pH gradient. We investigated the transient qE (qE(TR)) in different Arabidopsis (Arabidopsis thaliana) mutants. In dark-adapted plants, qE(TR) was absent in the npq4 mutant (deficient in the PsbS protein) and the pgr1 mutant (restricted in lumen acidification). In comparison with wild-type plants, qE(TR) was reduced in the zeaxanthin (Zx)-deficient npq1 mutant and increased in the Zx-accumulating npq2 mutant. After preillumination of plants (to allow the synthesis of large amounts of Zx), the formation and relaxation of qE(TR) was accelerated in all plants (except for npq4) in comparison with the respective dark-adapted plants. The extent of qE(TR), however, was unchanged in npq1 and npq4, decreased in npq2, but increased in wild-type and pgr1 plants. Even in presence of high levels of Zx, qE(TR) in pgr1 mutants was still lower than that in wild-type plants. In the presence of the uncoupler nigericin, qE(TR) was completely abolished in all genotypes. Thus, the transient qE(TR) shows essentially the same characteristics as the steady-state qE; it is strictly dependent on the PsbS protein and a low lumen pH, but the extent of qE(TR) is largely modulated by Zx. These results indicate that qE(TR) does not represent a different quenching mechanism in comparison with the steady-state qE, but simply reflects the response of qE to the dynamics of the lumen pH during light activation of photosynthesis.

Our FABulous VACation: a decade of phosphatidylinositol 3,5-bisphosphate

PtdIns(3,5)P2 was discovered about a decade ago and much of the machinery that makes, degrades and senses it has been uncovered. Despite this, we still lack a complete understanding of how the pieces fit together but some patterns are beginning to emerge. Molecular functions for PtdIns(3,5)P2 are also elusive, but the identification of effectors offers a way into some of these processes. An examination of the defects associated with loss of synthesis of PtdIns(3,5)P2 in lower and higher eukaryotes begins to suggest a unifying theme; this lipid regulates membrane retrieval via retrograde trafficking from distal compartments to organelles that are more proximal in the endocytic/lysosomal system. Another unifying theme is stress signalling to organelles, possibly both to change their morphology in response to external insults and to maintain the lumenal pH or membrane potential of organelles. The next few years seem likely to uncover details of the molecular mechanisms underlying the biology of this fascinating lipid. This review also highlights some areas where further research is needed.

Our FABulous VACation: a decade of phosphatidylinositol 3,5-bisphosphate

PtdIns(3,5)P2 was discovered about a decade ago and much of the machinery that makes, degrades and senses it has been uncovered. Despite this, we still lack a complete understanding of how the pieces fit together but some patterns are beginning to emerge. Molecular functions for PtdIns(3,5)P2 are also elusive, but the identification of effectors offers a way into some of these processes. An examination of the defects associated with loss of synthesis of PtdIns(3,5)P2 in lower and higher eukaryotes begins to suggest a unifying theme; this lipid regulates membrane retrieval via retrograde trafficking from distal compartments to organelles that are more proximal in the endocytic/lysosomal system. Another unifying theme is stress signalling to organelles, possibly both to change their morphology in response to external insults and to maintain the lumenal pH or membrane potential of organelles. The next few years seem likely to uncover details of the molecular mechanisms underlying the biology of this fascinating lipid. This review also highlights some areas where further research is needed.

Raising pH in the stomach lumen does not limit damage progression at the gastric epithelial surface following microscopic lesions

We have shown that during superfusion of the gastric surface with pH 3 solution, the response to microscopic (two-photon) photodamage includes rapid cell exfoliation and coincident epithelial repair. Our goal here is to ask if exfoliation is due to the stress of a highly acidic luminal pH. Methods: To measure surface pH, gastric mucosa of anesthetized mice was ratio imaged (Zeiss two-photon microscope) while the stomach was perfused with lightly buffered pH 3 saline plus Cl-NERF, or pH 7 saline plus BCECF. A 2-3 cell region of the epithelial surface (~ 225 μm2) was photodamaged by two-photon laser light (710nm, 0.4 W for 5-8 s). Photodamage was monitored through the loss of NAD(P)H autofluorescence, and cellular uptake of extracellular pH dye. Results: At pH 3, initial surface pH values averaged 3.08 (n=2), and reached maximum pH value of 3.66 after damage. At pH 7, initial pH values were 6.4 ± 0.3 (n=5), and reached maximum values of 6.6 ± 0.3 after damage. At pH 3 and pH 7, initially damaged areas were similar (90 μm2 at pH 3, n=2; 130 ± 124 μm2 at pH 7, n=4). Damage expanded to adjacent cells, resulting in a maximally damaged area of 1434 μm2 at pH 3 and 2115 ± 575 μm2 at pH 7. By 15 ± 10 min, cell exfoliation was complete at both luminal pH values. Conclusion: Expanding damage and exfoliation occur at widely variable luminal pH, suggesting the process is an intrinsic property of the gastric epithelium that does not require an acidic environment. Supported by NIH RO1 DK54940.