Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Measurement of Intracellular pH of Bullfrog Skeletal Muscle and Renal Tubular Cells with Double-Barreled Antimony Microelectrodes

A pencil-type antimony microelectrode of double-barreled design with a tip of less than 1 to 2 μm in outside diameter was constructed and used to measure intracellular pH(pHi) on frog sartorius muscle and renal tubular cells. Simultaneous observations of membrane potential difference (EM) were made. The results obtained were as follows: (1) The in vivo pHi of frog sartorius muscle was 7.12 ± 0.07 (SD) (n = 144); the simultaneously measured E_M was -51.1 ± 7.9 mV. The in vivo pHi of frog proximal tubule was 7.49 ± 0.07 (n = 221) and the E_M peri across the peritubular membrane was -50.2 ± 9.0 mV. (2) In proximal tubule in vivo, there was a negative correlation between pHi and E_M (r = -.62, p <. 05). On the other hand, in sartorius muscle in vivo, a positive correlation between the two was found (r =. 85, p <. 001). (3) In in vitro sartorius muscle, the pHi was 7.03 ± 0.14 (n = 9) and E_M was -62.4 ± 4.4 mV within one hour after isolation. (4) Increasing the external potassium concentration in the preparations to 75 mM caused a progressive depolarization by 43.3 ± 15.9 (m = 4) mV, while pHi changed in the alkaline direction by 0.22 ± 0.03 pH unit. (5) These results indicate that the pHi in both tissues does not obey the Donnan rule.     

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.     

Neuronal intracellular pH directly mediates nitric oxide‐induced programmed cell death

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH_i) regulation during nitric oxide (NO)‐induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick‐end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 μM), 3‐morpholinosydnonimine (300 μM), or 6‐(2‐hyrdroxy‐1‐methyl‐2‐nitrosohydrazino)‐N‐methyl‐1‐hexanamine (300 μM). The pH_i was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH_i 7.36 ± 0.01 to approximately 7.00 (p < .0001). Within 45 min, a biphasic alkaline response was evident, with pH_i reaching 7.40 ± 0.02, that was persistent for a 6‐h period. To mimic the effect of NO‐induced acidification, neurons were acid‐loaded with ammonium ions to yield a pH_i of 7.09 ± 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 ± 2% (24 h) and DNA fragmentation increased to 58 ± 8% (24 h) (p < .0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH_i of 7.60 ± 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO‐induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 171–184, 1999     

Cell pH and H+ Secretion by S3 Segment of Mammalian Kidney: Role of H+-ATPase and Cl−

The role of H⁺-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused ``in vitro' while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na⁺- and Cl⁻-free medium, cell pH fell by a mean of 0.37 ± 0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 ± 0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl⁻ channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na⁺ solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018 ± 0.0021 min⁻¹, not significantly different from zero (P > 0.42). For superfusion with Na⁺0 Ringer, this variation was 0.081 ± 0.015 min⁻¹, P < 0.001 against 0. These slopes were markedly reduced by the Cl⁻ channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na⁺-free medium is probably due to a vacuolar H⁺-ATPase, whose activity is stimulated in the presence of Cl⁻, and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl⁻ and on the integrity of the cytoskeleton. Received: 11 April 2000/Revised: 14 August 2000     

Activation of Na+/H+ exchange and Ca2+ mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization.

Although an increase in cytosolic pH (pHi) caused by Na+/H+ exchange enhances Ca2+ mobilization in platelets stimulated by low concentrations of thrombin [Siffert & Akkerman (1987) Nature (London) 325, 456-458], studies using fluorescent indicators for pHi (BCECF) and [Ca2+]i (fura2) suggest that Ca2+ is mobilized while the cytosolic pH decreases. Several lines of evidence indicate that the initial fall in BCECF fluorescence is not due to cytosolic acidification but is caused by a platelet shape change. (1) Pulse stimulation of platelets by successive addition of hirudin (4 unit/ml) and thrombin (0.2 unit/ml) induced a shape change of 43 +/- 8% and a fall in BCECF fluorescence, which both remained unchanged when Na+/H+ exchange was inhibited by ethylisopropylamiloride (EIPA, 100 microM). (2) Increasing the thrombin concentration to 0.4 unit/ml doubled the shape change and the fall in BCECF fluorescence, but again EIPA had no effect on these responses. (3) Treating platelets with 2 microM-ADP induced shape change and a decline in BCECF fluorescence that was unaffected by EIPA. (4) A second addition of thrombin to platelets that had already undergone shape change induced an immediate increase in BCECF fluorescence without a prior decrease. (5) Activation of protein kinase C by 1,2-dioctanoyl-sn-glycerol (DiC8) neither induced shape change nor a decline in BCECF fluorescence; in contrast BCECF fluorescence rapidly increased indicating an immediate cytosolic alkalinization. Concurrent analysis of [Ca2+]i under conditions in which shape change did not interfere with BCECF fluorescence showed that cytosolic alkalinization and Ca2+ mobilization started almost simultaneously. These observations suggest that cytosolic alkalinization is not preceded by a fall in pHi and can support Ca2+ mobilization induced by weak agonists.     

Comparison of interactions between human serum albumin and silver nanoparticles of different sizes using spectroscopic methods

Three different sizes (15.9 ± 2.1 nm, 26.4 ± 3.2 nm and 39.8 ± 4.0 nm, respectively) of citrate‐coated silver nanoparticles (SNPs) have been synthesized and characterized. The interactions of the synthesized SNPs with human serum albumin (HSA) at physiological pH have been systematically studied by UV‐vis absorption spectroscopy, fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The results indicate that the SNPs can bind to HSA with high affinity and quench the intrinsic fluorescence of HSA. The binding constants and quenching rate constants were calculated. The apparent association constants (K_app) values are 2.14 × 10⁴ M^–1 for 15.9 nm SNP, 1.65 × 10⁴ M^–1 for 26.4 nm SNP and 1.37 × 10⁴ M^–1 for 39.8 nm SNP, respectively. The values of binding constant obtained from the fluorescence quenching data match well with that determined from the absorption spectral changes. These results suggest that the smaller SNPs have stronger interactions to HSA than the larger ones at the same concentrations. Synchronous fluorescence, three‐dimensional fluorescence and CD spectroscopy studies show that the synthesized SNPs can induce slight conformational changes in HSA. Copyright © 2014 John Wiley & Sons, Ltd.     

A ratiometric fluorescence strategy based on copper nanoclusters/carbon dots for sensitive detection of doxorubicin

Sensitive detection of doxorubicin (DOX) is critical for clinical theranostics. A novel ratiometric fluorescence strategy based on the inner filter effect (IFE) has been established for the sensitive detection of DOX by designing a ratiometric fluorescence probe. In the presence of DOX, the fluorescence intensity of copper nanoclusters (CuNCs) at 485 nm decreases, and the fluorescence intensity of carbon dots at 560 nm increases. Therefore, DOX can be quantitatively detected by measuring the ratio of the fluorescence intensities at 560 and 485 nm (F₅₆₀/F₄₈₅). The F₅₆₀/F₄₈₅ ratio exhibits a linear correlation with the DOX concentration in the range from 1.0 × 10⁻⁸ M to 1.0 × 10⁻⁴ M with the detection limit of 3.7 nM. Furthermore, this method was also successfully applied to the analysis of DOX in human plasma samples, affording an effective platform for drug safety management.     

Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes

Three pH-sensitive fluorochromes-4-methyl-umbelliferone(4MU),2, 3-dicyano-hydroquinone (DCH), and 2',7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF)--were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore. Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.     

Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry

To monitor the specificity of Staphylococcus aureus aptamer (SA‐31) against its target cell, we used enzyme‐linked aptamer assay. In the presence of target cell, horseradish peroxidase–conjugated streptavidin bound to biotin‐labeled SA‐31 showed specific binding to S   aureus among 3 different bacteria with limit of detection of 10³ colony‐forming unit per milliliter. The apparent K _a was 1.39 μM⁻¹ ± 0.3 μM⁻¹. The binding of SA‐31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K _a, ΔH , and ΔG ). Since binding of aptamer to its targets solely depends on its 3‐dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA‐31 to its target on surface of bacteria. At 4°C, SA‐31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA‐31 slightly varied from K _a = 1.56 μM⁻¹ ± 0.69 μM⁻¹ at 25°C to K _a = 1.03 μM⁻¹ ± 0.9 μM⁻¹ at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S  aureus aptamer to its target were also 9.44 μM⁻¹ ± 0.38 μM⁻¹ at 50mM, 1.60 μM⁻¹ ± 0.11 μM⁻¹ at 137mM, and 3.28 μM⁻¹ ± 0.46 μM⁻¹ at 200 mM of salt concentration. In this study, it was demonstrated that enzyme‐linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S  aureus .     

H+ ATPase and Cl− Interaction in Regulation of MDCK Cell pH

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na⁺-H⁺ exchange, H⁺–K⁺ ATPase and Cl⁻/HCO⁻ ₃ exchange. In this work we studied the functional activity of a vacuolar H⁺-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H⁺ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH⁺ ₄ pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na⁺ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10⁻⁸ m (a specific inhibitor of the vacuolar H⁺-ATPase) was added to the zero Na⁺ solution. When using a solution with zero Na⁺, low K⁺ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H⁺–K⁺-ATPase. This result was confirmed by the use of 5 × 10⁻⁵ m Schering 28080. The Na⁺ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10⁻⁵ m (a specific blocker of Cl⁻ channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl⁻/normal Na⁺ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na⁺ and Cl⁻ free solution. From these results we conclude that: (i) MDCK cells have two Na⁺-independent mechanisms of pHi recovery, a concanamycin sensitive H⁺-ATPase and a K⁺ dependent, Schering 28080 sensitive H⁺–K⁺ ATPase; and, (ii) pHi recovery in Na⁺-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl⁻–free medium. This finding supports a role for chloride in the function of the H⁺ ATPase, which might be electrical shunting or a biochemical interaction. Received: 24 October 1997/Revised: 19 February 1998     

Binding of 4-methyl umbelliferyl-α-D-glucoPyranoside to Vicia faba lectin: Fluorescence-quenching studies

On binding toVicia faba lectin, the fluorescence of 4-methylumbelliferyl-α-D-glucoPyranoside was quantitatively quenched showing that the interaction of 4-methylumbelliferyl-α-D-glucoPyranoside took Place in a binding environment. The binding of the fluorescent sugar was saccharide sPecific as evidenced by the reversal of 4-methylumbelliferyl-α-D-glucoPyranoside fluorescence quenching by D-fructose. The association constant,K _a, values for the 4-methylumbelliferyl-α-D-glucoPyranoside was determined by comPetition study emPloying reversal of fluorescence quenching of 4-methylumbelliferyl-α-D-glucoPyranoside by D-fructose. TheK _a value obtained for D-fructose was 1.07 ±0.03 X 10⁴ M^-1 and for 4-methylumbelliferyl-α-D-glucoPyranoside was 1.60 ±0.05 X 10⁴ M^-1 at 15°C. TheK _a values of 2.51 ±0.06 X 10⁴M^-1, l.26 ±0.02 X 10⁴ M^-1 and 0.56 ±0.01 X 10⁴M^-1, resPectively at 10°, 20° and 30°C were obtained from the ChiPman equation. The relative fluorescence quenching, ΔF _a, at infinite concentration of the free saccharide sites ofVicia faba lectin [P′] was 93.5% at 30°C and the binding constant for 4-methylumbelliferyl-α-D-glucoPyranoside lectin interaction as derived by Yank and Hanaguchi equation was 0.63 ±0.01 X 10⁴M^-1.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot multiplication through shoot tip cultures of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., a threatened plant endemic to Southern India

Summary An efficient and rapid micropropagation system was developed for a food and medicinally important endangered shrub, Decalepis hamiltonii (‘swallow root’), through shoot multiplication. The influence of 2.5–7.5 μM isopentenyladenine (2iP), 4.4–17.7 μM 6-benzyladenine, 2.3–4.7 μM kinetin, 2.8–6.8 μM thidiazuron, and 2.3–11.4 μM zeatin alone and in combination with 0.3–0.9 μM indole-3-acetic acid (IAA) on in vitro multiple shoot production was studied. The maximum number of multiple shoots (6.5±0.4) was induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.9 μM 2iP. But, both zeatin (9.1 μM) and kinetin (4.7 μM) in combination with IAA (0.6 μM) were able to produce a maximum of 5.0±0.4 and 5.1±0.4 multiple shoots, respectively. Further elongation of shoots and adventitious shoot formation was obtained on medium containing 2.5 μM 2iP and 0.3 μM gibberellic acid. Elongated shoots were separated and rooted on MS medium supplemented with 9.8μM indole-3-butyric acid (IBA) and various phenolic compounds within 5–6 wk. Phloroglucinol and salicylic acid interaction with IBA stimulated in vitro rooting of shoots. Successful field transfer was achieved in rooted plantlets.     

Effects of different excitation and detection spectral regions on room temperature chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence parameters

The effects of different spectral region of excitation and detection of chlorophyll (Chl) a fluorescence at room temperature on the estimation of excitation energy utilization within photosystem (PS) 2 were studied in wild-type barley (Hordeum vulgare L. cv. Bonus) and its Chl b-less mutant chlorina f2 grown under low and high irradiances [100 and 1 000 μmol(photon) m⁻² s⁻¹]. Three measuring spectral regimes were applied using a PAM 101 fluorometer: (1) excitation in the red region (maximum at the wavelength of 649 nm) and detection in the far-red region beyond 710 nm, (2) excitation in the blue region (maximum at the wavelength of 461 nm) and detection beyond 710 nm, and (3) excitation in the blue region and detection in the red region (660– 710 nm). Non-photochemical quenching of maximal (NPQ) and minimal fluorescence (SV₀), determined by detecting Chl a fluorescence beyond 710 nm, were significantly higher for blue excitation as compared to red excitation. We suggest that this results from higher non-radiative dissipation of absorbed excitation energy within light-harvesting complexes of PS2 (LHC2) due to preferential excitation of LHC2 by blue radiation and from the lower contribution of PS1 emission to the detected fluorescence in the case of blue excitation. Detection of Chl a fluorescence originating preferentially from PS2 (i.e. in the range of 660–710 nm) led to pronounced increase of NPQ, SV₀, and the PS2 photochemical efficiencies (F_V/F_M and F_V′/F_M′), indicating considerable underestimation of these parameters using the standard set-up of PAM 101. Hence PS1 contribution to the minimal fluorescence level in the irradiance-adapted state may reach up to about 80 %.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Mechanism of cadmium-induced cytotoxicity in rat hepatocytes

Exposure of rat hepatocytes to cadmium below 50 μM for a short period (10 min) resulted in cellular acidification. Conversely, exposure to Cd more than 50 μM for a long period (60 min) caused cellular alkalinization accompanied by membrane damage as reflected by decrease in cellular K content and loss of intracellular lactic dehydrogenase. In hepatocytes exposed to 5 μM Cd, a concentration sufficient to induce acidification without cytotoxicity, the metal was preferentially associated with the crude nuclei and cell debris fractions, suggesting an interaction between Cd and cell membranes to cause acidification. Omission of bicarbonate from the incubation medium induced cellular acidification. The presence of Cd in this medium did not potentiate the medium-induced acidification. Mg-ATP (25 μM) induced cellular acidification in relation to an increase in the concentration of cytosolic free Ca. The coexistence of Mg-ATP and Cd at the concentrations which had no effect on cellular pH in the presence of either agants induced cellular acidification. These observations suggest that Cd induced cellular acidification by modulating the process connected with the rise in cytosolic free Ca via interaction with plasma membranes. This acidification had no strong immediate cytotoxic actions but led to subsequent cellular alkalinization accompanied with severe cytotoxicity and membrane breakage.     

Paper mill sludge-based carbon quantum dots as a specifically ratiometric fluorescent probe for the sensitive and selective detection of coptisine

Coptisine (COP), one of the bioactive components in Rhizoma Coptidis, has many pharmacological effects. Meanwhile, the determination of COP is essential in pharmacological and clinical applications. Herein, we prepared carbon quantum dots (CQDs) by one-step oil-thermal method using paper mill sludge (PMS) as precursor, and developed a ratiometric fluorescence method for the determination of COP. The structural and optical properties of PMS-CQDs were evaluated through high-resolution transmission electron microscopy (HRTEM), Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), ultraviolet-visible (UV-vis), fluorescence, zeta potential and fluorescence lifetime experiments. Fluorescence intensity ratio at 550 nm and 425 nm (I₅₅₀/I₄₂₅) was recorded as an index for quantitative detection of COP. The detection concentration of COP ranges from 0.1 to 50 μM in good linear correlation (R² = 0.9974) with a limit of detection of 0.028 μM (3σ/k). The quenching mechanism was deduced to be inner filter effect and static quenching. The ratiometric fluorescent probe showed impressive selectivity and sensitivity towards COP, and was successfully applied to the detection of COP in human urine with expected recoveries (95.22–111.00%) and relative standard deviations (0.46–2.95%), indicating that our developed method has a great application prospect in actual sample detection.     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.     

High-performance liquid chromatography separation and quantitation of ofloxacin enantiomers in rat microsomes

A sensitive, simple and accurate method for determination of enantiomers of ofloxacin in microsomal incubates was developed by chiral ligand-exchange RP-HPLC with fluorescence detection to examine stereoselective metabolism of ofloxacin in the glucuronidation process. The C₁₈ stationary phase was used as analytical column. The solution of chiral mobile phase additive was made up of 6 mM l-phenylalamine mixed with 3 mM CuSO₄ in water. Mobile phase consisted of the solution of chiral mobile phase additive–methanol (86:14).The fluorescence detector was operated at λ_ex 330 nm and λ_em 505 nm. The flow-rate of mobile phase was set at 1.0 ml/min. The achiral ODS column offers good separation of the two enantiomers in less than 25 min. The recovery of the assay was 97.9±6.1% (n=10) for S-ofloxacin and 99.6±6.0% (n=10) for R-ofloxacin. The method provides a high sensitivity and good precision (RSD<10%). The LOD was 0.6 μM for both enantiomers and the LOQ was 5.70±0.45 μM (n=8) for S-ofloxacin and 5.66±0.47 μM (n=8) for R-ofloxacin. The standard curves showed excellent linearity over the concentration range 5.5–2078 μM for S-(−)-ofloxacin and R-(+)-ofloxacin. The enantioselective method developed has been applied to determine the stereoselectivity of glucuronidation metabolism of ofloxacin optical isomers in rat liver microsomes.