P-31 in-vivo NMR investigation on the function of polyphosphates as phosphate-and energysource during the regreening of the green alga Chlorella fusca

The green alga Chlorella fusca accumulates polyphosphates under conditions of nitrogen starvation while deassembling the photosynthetic apparatus. The polyphosphate content of cells regreening after resupply with nitrate under different culture conditions was investigated by P-31 in-vivo NMR spectroscopy. Neither phosphate deficiency nor anaerobiosis during the first hours of regreening inhibited the recovery of the cells. Polyphosphates were degraded during regeening. Differences in the amount of polyphosphates of phosphate supplied and deficient cells occurred only after more then 8 h. After 16 h phosphate deficient cells had still 75% of the polyphosphate content of phosphate suppled cells. In cells kept under anaerobic conditions polyphosphate degradation was much higher than in oxygen supplied cells. After 8 h they contained less than 50% of the polyphosphate content of oxygen supplied cells. These data suggest that polyphosphates serve as obligatory phosphate source during regreening and may be used as an energy source.Non standard abbreviations EDTA Ethylene diamine tetraacetic acid - FID Free induction decay - MOPSO 3-(N-morpholine)-2-hydroxy-propanesulfonic acid - NMR Nuclear magnetic resonance - PP Polyphosphates - PP₄ central phosphate groups of polyphosphates     

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.     

Differential sensitivity of the cellular compartments of Saccharomyces cerevisiae to protonophoric uncoupler under fermentative and respiratory energy supply.

The effect of a protonophoric uncoupler (CCCP) on the different cellular compartments was investigated in yeast grown aerobically on lactate. These cells were incubated in a resting cell medium under three conditions; in aerobiosis with lactate or glucose or in anaerobiosis with glucose as energetic substrate. For each condition, in vivo 31P NMR was used to measure pH gradients across vacuolar and plasma membrane and phosphorylated compound levels. Respiratory rate (aerobic conditions) and TPP+ uptake were measured independently. Concerning the polyphosphate metabolism, spontaneous NMR-detected polyphosphate breakdown occurred, in anaerobiosis and in the absence of CCCP. In contrast, in aerobiosis, polyphosphate hydrolysis was induced by addition of either CCCP or a vacuolar membrane ATPase-specific inhibitor, bafilomycin A1. Moreover, polyphosphates were totally absent in a null vacuolar ATPase activity mutant. The vacuolar polyphosphate content depended on two factors: vacuolar pH value, strictly linked to the vacuolar H(+)-ATPase activity, and inorganic phosphate concentration. CCCP was more efficient in dissipating the proton electrochemical gradient across vacuolar and mitochondrial membranes than across the plasma membrane. This discrepancy can be essentially explained by a difference of stimulability of each proton pump involved. As long as the energetic state (measured by NDP + NTP content) remains high, the plasma membrane proton ATPase is able to compensate the proton leak. Moreover, this ATPase contributes only partially to the generation of delta pH. The maintenance of the delta pH across the plasma membrane, that of the energetic state, and the cellular TPP+ uptake depend on the nature of the ATP-producing process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of lysosomal and cytosolic pH in the regulation of macrophage lysosomal enzyme secretion.

Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH.     

Growth kinetics of Schizosaccharomyces pombe under various culture conditions: influence of pH,malate, ethanol and oxygenation

Summary The effect of various culture conditions on growth kinetics of a Schizosaccharomyces pombe strain were investigated in order to facilitate the potential use of this organism in enology. The effect of ethanol was characterized as a 80 g·l^–1 critical concentration. An optimal pH range of 3.5–4.0 was found. Growth was slightly affected by malic acid at pH 3.0 but was shown to decrease drastically at pH 5.5. Aerated cultures had increased rates of growth and malate degradation than those grown under anaerobiosis, although the specific rate of malate degradation remained fairly constant. Taking into account biomass synthesis data it would seem likely that under anaerobiosis malate can replenish the Krebs' cycle for amphibolic precursor synthesis and hence avoid potential growth limitation. Offprint requests to: A. Pareilleux     

Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator.

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.     

Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L.) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst. Received: 23 November 1998 / Accepted: 23 June 1999     

Vesicular ATPase inserted into the plasma membrane of motor terminals by exocytosis alkalinizes cytosolic pH and facilitates endocytosis

Key components of vesicular neurotransmitter release, such as Ca(2+) influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca(2+) influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (～ 13 nM average peak increase in [H(+)]), followed by a prolonged alkalinization (～ 30 nM peak decrease in [H(+)]) that outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H(+) pumping by vesicular H(+)-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H(+)-ATPase, known to transfer cytosolic H(+) into prefused vesicles, continues to extrude cytosolic H(+) after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.     

Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.     

Heparin requirement for the quantitation of fibrinogen production by primary hepatocyte cultures

We have reported a rapid method for the quantitation of proteins secreted in culture media ([12.]). Using the same method, we observe that serum-free rat hepatocyte cultures exhibited a 100% increase in detectable secreted fibrinogen-antigen in the presence of 1 unit/ml heparin or greater at 24 h of culture. The amount of transferrin, haptoglobin, and albumin detected was unaltered by the presence of heparin. Since heparin is known to affect certain cellular functions, the fates of [³⁵S]methonine-labeled fibrinogen in cell extracts and culture media were examined employing pulse-chase experiments. Labeled intracellular fibrinogen disappeared at similar rates and was initially released into the media in similar amounts in the presence or absence of heparin. At 8 h during the chase, there was a 40–50% reduction in fibrinogen-antigen in spent culture medium lacking heparin. The presence of heparin did not alter the proteolytic degradation of secreted fibrinogen as determined by immunoblotting of spent culture media proteins separated by polyacrylamide gel electrophoresis. In vitro experiments indicate that clotting of fibrinogen by thrombin reduces the amount of immunodetectable fibrinogen. The results indicate that heparin increases the amount of detectable fibrinogen secreted by cultured hepatocytes by preventing clotting and not by stimulating synthesis or secretion or by inhibiting degradation. Hence, it is critically important to include heparin when secreted fibrinogen is quantitated by the method that we have developed.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2,7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of ¹⁴C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25–37.5 mm in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO ₃ ^– , both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A₁ caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO₂) was sensitive to both 25 mm NO ₃ ^– and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5–1.0 mm ouabain resulted in an initial alkalinization (0.5–2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mm NO ₃ ^– , but not by 25 mm gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H⁺ uptake by V-ATPase activity. We conclude that V-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.This work was supported in part by National Institutes of Health B.R.S. Grant 507 RR05417 to Temple University.     

The synthesis of proteoglycans by human T lymphocytes

We have examined the proteoglycans produced by highly-purified cultures of human T-lymphocytes. The proteoglycans were metabolically labelled with [35S]sulphate and analysed in cellular and medium fractions using DEAE-cellulose chromatography, gel filtration and specific enzymatic and chemical degradations. The results showed that the T cells synthesized a relatively homogeneous, proteinase-resistant chondroitin 4-sulphate proteoglycan that accumulated in the culture medium during a 48 h incubation period. The cellular fraction contained a significant amount of free chondroitin sulphate chains that were not secreted into the medium. These polysaccharides were formed by intracellular degradation of proteoglycan in a chloroquine-sensitive process, indicating a requirement for an acidic environment. In contrast to chondroitin sulphate derived from proteoglycan, chondroitin sulphates synthesized on the exogenous primer, beta-D-xyloside, were mainly secreted by the cells. beta-D-Xylosides caused an 8-fold stimulation in the synthesis of chondroitin sulphate, but decreased the synthesis of proteoglycan by about 50%. These proteoglycans contained shorter chondroitin sulphate chains than their normal counterparts. The results indicate that although proteoglycans are mainly secretory components in human T-cell cultures, a specific metabolic step leads to the intracellular accumulation of free glycosaminoglycans. Separate functions are likely to be associated with the intracellular and secretory pools of chondroitin sulphate.     

Phosphorus-31 nuclear magnetic resonance investigation of the in vivo regulation of intracellular pH in cell suspension cultures of Nicotiana tabacum: the effects of oxygen supply, nitrogen, and external pH change

31P NMR was used to study the in vivo response of intracellular phosphorus-containing compounds of cell suspension cultures of Nicotiana tabacum to extracellular events. Limitation of the oxygen supply in a static system (sealed tube) caused a strong pH decrease of the cytoplasm and a smaller fall in the vacuolar pH. The rate of this process was independent of the age of the cells, except for those at the end of the growth cycle where either reserve supplies of inorganic phosphate have been depleted or metabolism has ceased. The cells can be returned quickly to their normal pH and metabolic status by reoxygenation after depletion times as long as 4.5 h. Regassing with N2 after anaerobiosis also caused the return of the nearly normal pH status, which indicates that rapid cell acidification is caused almost entirely by CO2 accumulation. In a second type of anaerobiosis, attained by continual passage of N2, cytoplasmic pH fell only slowly to a constant value higher than in the static case. Here acidification appeared to arise, at least in part, from lactate accumulation. Provided aeration occurred, the cytoplasm was maintained at a constant pH of 7.5 +/- 0.1 for changes in the medium pH value between 6.5 and 3. The biochemical and biotechnological implications of these results are discussed.     

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins.

We observed that wild-type Escherichia coli utilized a linear polyphosphate with a chain length of 100 phosphate residues (poly-P100) as the sole source of phosphate in growth medium. A mutation in the gene phoA of alkaline phosphatase or phoB, the positive regulatory gene, prevented growth in this medium. Since no alkaline phosphatase activity was detected outside the wild-type cells, the periplasmic presence of the enzyme was necessary for the degradation of polyphosphate. A 90% reduction in the activity of periplasmic acid phosphatase with a pH optimum of 2.5 (delta appA mutants) did not affect polyphosphate utilization. Of the porins analyzed (OmpC, OmpF, and PhoE), the phoB-inducible porin PhoE was not essential since its absence did not prevent growth. To study how poly-P100 diffused into the cells, we used high-resolution 31P nuclear magnetic resonance (31P NMR) spectroscopy. The results suggest that poly-P100 entered the periplasm and remained in equilibrium between the periplasm and the medium. When present individually, porins PhoE and OmpF facilitated a higher permeability for poly-P100 than porin OmpC did. The degradation of polyphosphate by intact cells of E. coli observed by 31P NMR showed a time-dependent increase in cellular phosphate and a decrease in polyphosphate concentration.     

Uptake of magnesium,strontium, barium,and manganese byPlectonema boryanum (Cyanophyceae) with special reference to polyphosphate bodies

Summary The ability of a cyanobacterium to take up and concentrate metals in polyphosphate bodies was tested. It was found that Mg, Ba and Mn are taken up by 3 and 4 week old cultures ofP. boryanum and in all cases the metals were concentrated in the polyphosphate bodies. In the case of Mn it was also detected in the cytoplasm. Strontium was sometimes taken up and incorporated into dense bodies which contained Sr, high S, high K and low Ca. In a number of trials no uptake of Sr could be detected. Polyphosphate bodies, formed as a result of the overplus phenomenon did not incorporate the metal at the time they are being formed. Polyphosphate bodies from 2 and 3 week culture contain P, K, Ca and Mg while bodies from 4 week cultures also possess Zn.It is suggested that sequestering metals in polyphosphate bodies may be a significant mechanism by which heavy metals are concentrated.     

The diacylglycerol analogue, 1,2-sn-dioctanoylglycerol, induces an increase in cytosolic free Ca2+ and cytosolic acidification of T lymphocytes through a protein kinase C-independent process.

In this paper, we demonstrate that low concentrations (0.5-2.5 microM) of 1,2-sn-dioctanoylglycerol (DiC8), a potent diacylglycerol used in many previous studies to probe the role of protein kinase C (PKC) in cell activation, cause cytosolic alkalinization of human, mouse and pig T lymphocytes through PKC-mediated activation of the Na+/H+ antiport. However, at higher concentrations (greater than or equal to 12.5 microM), the effect on cytosolic pH (pHi) is reversed, resulting in a marked cytosolic acidification, followed by a gradual return of pHi to baseline values. DiC8 also induces marked changes in cytosolic free calcium concentrations ([Ca2+]i), initially by releasing calcium from intracellular stores, followed by a net transmembrane influx of calcium. The DiC8-induced cytosolic acidification, the resultant return to baseline pH and the increase in [Ca2+]i are independent of activation of PKC. Unlike many other agents which increase [Ca2+]i, DiC8 does not induce phosphatidylinositol hydrolysis with the resultant production of inositol phosphates. Other compounds known to activate PKC, including the closely related diacylglycerol analogues, 1,2-sn-dihexanoylglycerol and 1,2-sn-didecanoylglycerol, phorbol esters and mezerein, did not induce changes in [Ca2+]i or cytosolic acidification in T lymphocytes. Thus the action of DiC8 on intact lymphocytes is different from that of phorbol esters and other diacylglycerols, and is specific to the length of the acyl chains. Because changes in [Ca2+]i are often associated with cell proliferation and cell differentiation, some effects of DiC8 on intact cells may be a consequence of changes in [Ca2+]i.     

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.     

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Photodegradation of Fleroxacin Injection: II. Kinetics and Toxicological Evaluation

Photodegradation kinetics of fleroxacin were investigated in different injections. Five commercial formulations were analyzed before and after irradiation by determining residual volumes of fleroxacin with high-pressure liquid chromatography (HPLC), and different decomposition functions and models were obtained. Concentration levels of fleroxacin in injections caused the differences in photodegradation kinetics instead of ingredients. Influences of different pH values and presence of NaCl on photodegradation of fleroxacin were observed. Low pH value decreased the efficacy of photolysis and enhanced photostability of fleroxacin injections. Tentative structure of a new degradation product afforded was proposed. An acute toxicity assay using the bioluminescent bacterium Q67 was performed for fleroxacin injections after exposure to light. The research proved that fleroxacin was more photolabile in dilute injection, and acute toxicity of dilute injection increased more rapidly than that of concentrated injection during irradiation.