pH-dependent effects of the ionophore nigericin on response of mammalian cells to radiation and heat treatment

The extracellular pH (pHe) in many solid tumors is often lower than the pH of normal tissues. The K+/H+ ionophore nigericin is toxic to CHO cells when pHe is below but not above 6.5, and thus it has potential for selective killing of tumor cells in an acidic environment. This study examines the pH-dependent effects of nigericin on the response of CHO cells to radiation and heat treatment. Cells held for 4 h in Hank's balanced salt solution, after 9 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is maximal at pHe 6.7-6.8. Addition of nigericin, postirradiation, not only inhibits PLDR when pHe is below 6.8, but interacts synergistically with radiation to reduce survival below that of cells plated immediately after irradiation when pHe is 6.4 or lower. Nigericin enhances heat killing of CHO cells perferentially under acidic conditions, and where neither heat nor drug treatment alone is significantly toxic. Survival of cells held for 30 min at 42.1 degrees C in the presence of 1.0 microgram/ml nigericin is 0.6, 0.08, 0.003, and 0.00003 at pHe 7.4, 6.8, 6.6, and 6.4, respectively, relative to survival of 1.0 in untreated cultures. The biochemical effects of nigericin at pHe 7.4 vs pHe 6.4 have been investigated. Nigericin inhibits respiration, stimulates glucose consumption, and causes dramatic changes in intracellular concentrations of Na+ and K+ at pHe 7.4 as well as 6.4. The drug reduces intracellular levels of ATP, GTP, and ADP but has more pronounced effects under acidic incubation conditions. Others have shown that nigericin equilibrates pHe and intracellular pH (pHi) only when pHe is 6.5 or lower. Our observations and those of others have led us to conclude that lowering of pHi by nigericin is either the direct or indirect cause of enhancement of radiation and heat killing of cells in an acidic environment.     

The role of low intracellular or extracellular pH in sensitization to hyperthermia

Cells are more sensitive to heat when they are heated in an acidic environment, and this study confirms (K. G. Hofer and N. F. Mivechi, J. Natl. Cancer Inst., 65, 621, 1980) that intracellular pH (pHi) and not extracellular pH (pHe) is responsible for the sensitization. The relationship between pHe, pHi, and heat survival of cells heated in vitro in various buffers at pHe 6.3-8.0 was investigated. Cells' adaptation to low environmental pH in terms of increases in pHi and heat survival also was investigated. Finally, we studied the relationships among pHe, pHi, and survival from heat for cells heated in sodium-free reconstructed medium. Intracellular pH was measured by the distribution of the weak acid, [2-14C]5,5-dimethyl-2,4-oxazolidinedione. Our results are summarized as follows: (1) CHO cells maintained the same relationship between pHe and pHi in four different media or buffers (McCoy's 5a medium buffered with CO2 and NaHCO3 or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) and 2-(N-morpholino)ethanesulfonic acid (Mes), Krebs-Ringer bicarbonate solution, and Krebs-Ringer phosphate solution) with pHi being 0.05 to 0.20 pH units higher than pHe as it varied from 7.0 to 6.4; furthermore, heat sensitization by acid was the same in medium buffered with NaHCO3 or Hepes and Mes. (2) The low pHe adapted cells multiplied with an increased doubling time of 20.7 +/- 0.7 h and appeared morphologically similar to the unadapted cells. However, the pHi of these cells was 0.15-0.30 pH units higher than that of the unadapted cells when pHe was varied between 7.0 and 6.3. (3) After being heated at 43.5 degrees C for 55 min or at 42.5 degrees C for 150 min at pHe 6.3-7.2, the pHi of the adapted cells increased by 0.2-0.1 pH units. However, heat caused no significant change in the unadapted cells. (4) Heat survival plotted versus pHe was 1000-fold higher for the adapted cells than for the unadapted cells at pHe of 6.3. However, heat survival plotted versus pHi was identical for the two cell types. (5) In sodium-free reconstructed McCoy's 5a medium, pHi was 0.25-0.1 pH units lower than that in the sodium-containing counterpart at pHe 6.3-7.2, and heat sensitization increased accordingly; however, heat survival plotted versus pHi was identical for the two types of media.     

Electrogenerated chemiluminescence of tris(2‐phenylpyridine)iridium(III) in water,acetonitrile and trifluorethanol

The spectroscopic, electrochemical and coreactant electrogenerated chemiluminescence (ECL) properties of Ir(ppy)3 (where ppy = 2‐phenylpyridine) have been obtained in aqueous buffered (KH2PO4), 50 : 50 (v/v) acetonitrile–aqueous buffered (MeCN–KH2PO4) and 30% trifluoroethanol (TFE) solutions. Tri‐n‐propylamine was used as the oxidative‐reductive ECL coreactant. The photoluminescence (PL) efficiency (ϕem) of Ir(ppy)3 in TFE (ϕem ≈ 0.029) was slightly higher than in 50 : 50 MeCN–KH2PO4 (ϕem ≈ 0.0021) and water (ϕem ≈ 0.00016) compared to a Ru(bpy)32+ standard solution in water (Φem ≈ 0.042). PL and ECL emission spectra were nearly identical in all three solvents, with dual emission maxima at 510 and 530 nm. The similarity between the ECL and PL spectra indicate that the same excited state is probably formed in both experiments. ECL efficiencies (ϕecl) in 30% TFE solution (ϕecl = 0.0098) were higher than aqueous solution (ϕecl = 0.00092) system yet lower than a 50% MeCN–KH2PO4 solution (ϕecl = 0.0091). Copyright © 2014 John Wiley & Sons, Ltd.     

Superoxide dismutase activity of human plasma; the effect of Cu2+-complex compounds

The superoxide-dismutase (EC 1.15.1.1) activity was revealed in blood plasma after its successive treatment with alcohol, chloroform and one- or two-substituted phosphate. Its value is the highest when treating plasma with KH2PO4. The boiling of the supernatant liquid obtained after addition of K2HPO4 leads to a complete loss of the activity. The treatment of plasma with KH2PO4 provides higher stability of the enzyme to thermal actions. The analysis of EPR-spectra has shown the presence of different complex compounds of copper ions in the supernatant liquid. Certain experiments with the use of aqueous solutions of CuCl2 and histidine have shown that Cu2+ inhibits the superoxide-dismutase activity in samples treated with KH2PO4 and increases it in samples treated with K2HPO4.     

Flow cytometric measurement of intracellular pH in B16 tumors: intercell variance and effects of pretreatment with glucose

Flow cytometry was used to measure cytoplasmic pH (pHi) of B16 melanoma cells taken from tumor-bearing animals. We used a ratiometric method to allow measurements on an individual cell basis which were independent of cellular content of the pH indicator BCECF. In order to "freeze" any intercell variance which may have existed within the tumor mass, tumors were mechanically disaggregated in bicarbonate-free medium containing 0.5 mM amiloride at 4 degrees C and loaded with BCECF in choline chloride-based Earle's solution at 37 degrees C. Studies using cells grown in vitro showed that this protocol prevented acid load recovery during the 30-min period typically required between tumor excision and pHi measurement. A calibration curve was obtained by resuspending BCECF-stained cells in a range of buffers containing the proton ionophore nigericin. The range of values for individual cells was estimated by comparing the coefficient of variation of the test sample with that obtained when nigericin was used to reduce all cells to the pHi of the calibration buffer. The average value for mean tumor cell pH was 7.32 +/- 0.05 SD. Pretreatment of animals with intraperitoneal glucose for one hour resulted in an average for mean pHi of 7.17 +/- 0.17 SD. Mean coefficient of variation was 8.7%, and in the presence of nigericin, 8.1%. These values indicate a variance in measured pHi of approximately +/- 0.4 pH units, but most of this results from experimental error rather than true intercell pHi variance. The method used here is capable of detecting reduction in mean tumour pHi caused by ip glucose, but incapable of precise estimation of individual cell values. Despite these uncertainties, the results suggest that the range of pHi within B 16 tumors is small.     

基于钙黄绿素-铜（Ⅱ）荧光体系测定乙酰半胱氨酸

目的：基于钙黄绿素-铜（Ⅱ）荧光体系测定乙酰半胱氨酸。方法：在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,以492 nm为激发波长,520 nm为发射波长测定乙酰半胱氨酸溶液的荧光强度。结果：在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,二价铜离子与钙黄绿素配位引起荧光猝灭。由于乙酰半胱氨酸中巯基上的硫离子与Cu2＋的亲和力很强,可从钙黄绿素-铜（Ⅱ）的络合物中夺取铜离子而使钙黄绿素游离出来,从而使体系的荧光得以恢复,并且荧光恢复的程度与加入乙酰半胱氨酸的量在一定范围内成线性。结论：建立了一种测定乙酰半胱氨酸的荧光分析新方法,该方法的线性范围为6.0 10-6～1.4 10-5 mol/L,检出限为4.010-6 mol/L。     

Membrane potential and cation content of osteoblast-like cells (UMR 106) assessed by fluorescent dyes

A number of cellular functions have recently been associated with alterations of the membrane potential in non-excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage-sensitive oxonol dye di-BA-C4(3) was developed. The fluorescent signal of di-BA-C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH-sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the "putative" rat osteoblasts (UMR 106) as -28.3 +/- 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 +/- 0.7 mM (n = 3) and 24 +/- 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.     

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium

BACKGROUND: The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. METHOD/PRINCIPAL FINDINGS: Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. CONCLUSIONS: The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.     

Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

BACKGROUND: The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. METHODS: To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. RESULTS: The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. CONCLUSIONS: The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.     

Intracellular pH (pHi) of red cells stored in acid citrate dextrose medium. Effects of temperature and citrate anions.

The intracellular pH (pHi) of red cells stored in acid citrate dextrose (ACD) medium was estimated by the 5,5'-dimethyloxazoldine,-2,4-dione (DMO) method. The initial pHi at 4degrees was about 7.6 and was higher than the extracellular pH (pHe) at 4degrees. During storage, both pHi and pHe decreased, but the former was always higher than the latter and the former decreased more slowly than the latter. The high pHi of ACD blood was a results of the temperature at which the pHe and the pHi were measured (4degrees) and the presence of citrate anions in the medium, and could be explained by application of the Donnan-Gibbs equilibrium. ATP and 2,3-diphosphoglycerate (DPG) were well-maintained in heparinized blood when it was acidified and pHe and pHi at 4degrees were both about 7.4, which suggests that improvement of blood preservation may be attained by suitable adjustment of the pHi and pHe of the blood.     

High phosphate (up to 600 mM) induces pseudohyphal development in five wild type Candida albicans

A method is described for the formation of nearly 100% pseudohyphae populations of wild-type Candida albicans A72. The method employs fungal growth at 37 degrees C (ca. 5x10(6) cells/ml) in a glucose-proline-N-acetyl-glucosamine medium supplemented with up to 600 mM phosphate (KH(2)PO(4)/K(2)HPO(4) 1:1) at pH 6.5. Four other strains of C. albicans (MEN, 10261, SG5314 and CAI-4) also formed pseudohyphae under these conditions, although the phosphate response profiles differed in the concentration required for each strain to form pseudohyphae.     

Production of Galacto-Oligosaccharide from Lactose by Sterigmatomyces elviae CBS8119

Volume 61, no. 11, p. 4022, column 2, line 10: "K(inf2)HSO(inf4)" and "KH(inf2)SO(inf4)" should read "K(inf2)HPO(inf4)" and "KH(inf2)PO(inf4)," respectively. Line 13: "K(inf2)HSO(inf4)" should read "K(inf2)HPO(inf4)." Line 14: "KH(inf2)SO(inf4)" and "0.2 g of CaCO(inf3)" should read "KH(inf2)PO(inf4)" and "1 g of CaCO(inf3)," respectively. [This corrects the article on p. 4022 in vol. 61.].     

The effect of the anions, phosphate and sulphate, on normal rates of excretion of potassium in dogs.

Small doses of (NH4)2HPO4 or KH2PO4 by stomach tube caused increase in plasma PO4 and PO4 excretion. Above a threshold of 0-8 mmol. 1(-1), increase of plasma PO4 by 0-5 mmol. 1(-1) caused PO4 excretion to increase by about 35 mumol. min.-1 After KH2PO4 this relationship was not altered by the concurrent increases in plasma K and K excretion. After doses of (NH4)2SO4 or K2SO4, excretion of SO4 was similarly related to plasma SO4 and was independent of plasma K and K excretion. An effect of PO4 on K excretion was observed after doses of (NH4)2HPO4, when increased excretion of PO4 was accompanied by increased excretion of K without change in plasma K. There was also increased excretion of NH4 and a small increase in Na excretion. The changes were similar to those produced by (NH4)2SO4 [O'Connor and Summerill, 1976]. KH2PO4 and K2SO4 produced increase in plasma K and increased excretion of K not significantly different from the changes produced by KCl or KHCO3 [Baylis and O'Connor, 1976]. After KH2PO2 or K2SO4, the urinary anion was PO4 or SO4, instead of Cl and HCO3. Any effect of anions on K excretion was much less than the effect of increase in plasma K. At low rates of excretion of K, increased urinary excretion of impermeant anion can determine increased excretion of K. However, the effect of anion is small in comparison with the effect of increase in plasma K.     

Buffer power and intracellular pH of frog sartorius muscle.

Intracellular pH (pHi) and buffer power of frog muscle were measured using pH-sensitive microelectrodes under conditions used previously in energy balance experiments because pH strongly influences the molar enthalpy change for phosphocreatine splitting, the major net reaction during brief contractions. The extracellular pH (pHe) of HEPES buffered Ringer's solution influenced pHi, but change in pHi developed slowly. Addition or removal of CO2 or NH3 from the extracellular solution caused a rapid change in pHi. The mean buffer power measured with CO2 was 38.4 mmol.l-1.pH unit-1 (+/- SEM 2.1, n = 49) and with NH3 was 36.2 (+/- SEM 5.5, n = 4) at 20-22 degrees C. At 5 degrees C, in experiments with CO2 the mean buffer power was 40.3 (+/- SEM 2.6, n = 3). For pHi values above approximately 7.0, the observed buffer power was greater than that expected from the values in the literature for the histidine content of intracellular proteins, carnosine and inorganic phosphate in the sarcoplasm. The measured pHi values were similar to those assumed in energy balance calculations, but the high measured buffer power suggests that other buffering reactions occur in addition to those included in energy balance calculations.     

Direct measurement of intracellular pH in identified glial cells and neurones of the leech central nervous system

Neutral carrier pH-sensitive double-barrelled microelectrodes were used to investigate intracellular pH (pHi) in leech neuropile glial cells and in Retzius neurones. The mean pHi of the glial cells was 6.87 +/- 0.13 (+/- SD, n = 27) in HEPES-buffered saline (pHo 7.4) and 7.18 +/- 0.19 (n = 13) in solutions buffered with 2% CO2- 11 mM HCO3-. The distribution of H+ ions in both the glia and neurones was found not to be in electrochemical equilibrium. To investigate pHi regulation, the pHi was decreased by exposure to CO2 or by adding and then removing NH4Cl. Acidification by any method was followed by a recovery to normal pHi values within minutes. The pHi recovery from acidification in neuropile glial cells in HEPES-buffered saline and CO2-HCO3- buffered saline was, however, blocked by removing external Na. In HCO3(-)-free solutions the diuretic amiloride (2 mM) reduced the rate of pHi recovery. In the presence of HCO3-, the rate of acid efflux was stimulated; the stilbene 4-acetamido-4'-isothiocyanatostilbene-2,3'-disulfonic acid (SITS; 0.5 mM) slowed pHi recovery. In HEPES buffered and CO2-HCO3- buffered solutions pHi regulation in neurones was inhibited by removing external Na. In HCO3(-)-free solutions amiloride reduced the rate of pHi recovery considerably. In the presence of HCO3-, SITS or amiloride slowed but did not completely block pHi recovery. We conclude that leech glial cells and neurones have two mechanisms of pHi regulation, one being Na+-H+ exchange and the other Na+ and HCO3- dependent.     

The role of low intracellular or extracellular pH in sensitization to hyperthermic radiosensitization

Previous work showed that intracellular pH (pHi) and not extracellular pH (pHe) was the determinant in the low pH sensitization of hyperthermic killing. The present studies show that the same is true for heat-induced radiosensitization and loss of cellular DNA polymerase activities. Chinese hamster ovary cells after they had adapted to low pH (6.7) had an increase in pHi which rendered cells partially resistant to the low pH sensitization of heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities. These results were quantified by plotting versus pHe, both the thermal enhancement ratio (TER), defined as the ratio of the X-ray dose without heat to the X-ray dose with heat to give an isosurvival value of 0.01, and the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to the D0 of the radiation survival curve for heat plus radiation. Both the TER and TEF were higher for the unadapted cells than for the adapted cells, i.e., 1.3-1.4 fold higher at a pHe of 6.3. However, the TER or TEF plotted versus pHi was identical for the two cell types. Finally, heat-induced loss of cellular DNA polymerase activities correlated with pHi and not pHe. Therefore, we conclude that pHi and not pHe is responsible for the increase by acid in heat-induced radio-sensitization and loss of cellular DNA polymerase activities.     

Role of monocarboxylic acid transport in intracellular pH regulation of isolated proximal cells

Isolated proximal cells were prepared from rabbit kidney cortex by mechanical dissociation. The intracytoplasmic pH (pHi) was measured in HCO3(-)-free media (external pH (pHe), 7.3) using the fluorescent dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). Cells were acid-loaded by the nigericin technique. Addition of 70 mM Na+ to the cells caused a rapid pHi recovery, which was blocked by 0.5 mM amiloride. When the cells were exposed to 5 mM sodium butyrate in the presence of 1 mM amiloride, the H+ efflux was significantly increased and followed Michaelis-Menten kinetics. Increasing pHe from 6.4 to 7.6 at a constant pHi of 6.4 enhanced the butyrate activation of the H+ efflux. Increasing pHi from 6.5 to 7.2 at a constant pHe of 7.2 reduced the butyrate effect. 22Na uptake experiments in the presence of 1 mM amiloride showed that 1.5 mM butyrate increased the Na+ flux in the proximal cells (pHi 7.10). The efficiency of monocarboxylic anions in promoting a pHi recovery increased with the length of their straight chain (acetate less than propionate less than butyrate less than valerate). The data show that when the Na+/H+ antiporter is blocked, the proximal cells can regulate their pHi by a Na+-coupled absorption of butyrate followed by non-ionic diffusion of butyric acid out of the cell and probably also by OH- influx by means of the OH-/anion exchanger.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

Measurement of intracellular pH in hamster diaphragm by absorption spectrophotometry

The regulation of intracellular pH (pHi) is important in controlling muscle contraction. In these experiments, a spectrophotometric method of determining pHi was developed, and the method was then used to study muscle pHi regulation during CO2-induced changes in extracellular pH (pHb). Studies were performed in vitro on 27 diaphragm muscle strips obtained from adult hamsters. pHi was measured from the ratio of the absorbances of the acid (lambda = 530 nm) and alkaline (lambda = 460 nm) forms of a vital dye, neutral red, using the unstained diaphragm spectrum as a reference blank. A standard neutral red calibration curve constructed from eight diaphragm muscle homogenates indicated that the absorbance ratio was highly linear, with pH over the range 6.00-8.00. In intact muscle strips gassed with 95% O2-5% CO2, pHb was 7.45 +/- 0.03 (SE) and pHi was 7.00 +/- 0.01 (SE). When the muscle was aerated with CO2 concentrations from 3 to 30%, pHb and pHi changed rapidly and reached a steady state in 10-15 min. However, when pHb ranged from 6.80-7.80, pHi changed little from the value observed when pHb was 7.40. When pHb was less than 6.80 or greater than 7.80, changes in pHi and pHb were quantitatively similar. The results suggest that, in the isolated diaphragm, overall pHi is stable and effectively buffered over a wide range of CO2-induced changes in buffer solution pH.     

Application of statistical experimental designs for the optimization of medium constituents for the production of citric acid from pineapple waste

Statistical experimental designs were applied for the optimization of medium constituents for citric acid production by Yarrowia lipolytica NCIM 3589 in solid state fermentation (SSF) using pineapple waste as the sole substrate. Using Plackett-Burman design, yeast extract, moisture content of the substrate, KH(2)PO(4) and Na(2)HPO(4) were identified as significant variables which highly influenced citric acid production and these variables were subsequently optimized using a central composite design (CCD). The optimum conditions were found to be yeast extract 0.34 (%w/w), moisture content of the substrate 70.71 (%), KH(2)PO(4) 0.64 (%w/w) and Na(2)HPO(4) 0.69 (%w/w). Citric acid production at these optimum conditions was 202.35 g/kg ds (g citric acid produced/kg of dried pineapple waste as substrate).