Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

Ecology of aquatic Ranunculus communities under the Mediterranean climate

The Iberian Peninsula encompasses more than 80% of the species richness of European aquatic ranunculi. The floristic diversity of the phytocoenosis characterised by aquatic Ranunculus and the main physical–chemical factors of the water were studied in 43 localities of the central Iberian Peninsula. Four aquatic Ranunculus communities are found in most of the aquatic environments. These are species-poor and have an uneven distribution: three species of Batrachium are heterophyllous and their communities are distributed in different aquatic ecosystems on silicated substrates; one species is homophyllous and its community occurs in various aquatic ecosystems with carbonated waters. In the Mediterranean climate, Ranunculus species are present in different habitats, as shown by the results of all the statistical analyses. Ranunculus trichophyllus communities occur in base-rich waters with a high buffering capacity (2273.44 ± 794.57 mg CaCO₃ L⁻¹) and a high concentration of cations (Ca²⁺, 121 ± 33.12 mg L⁻¹; Mg²⁺, 71.64 ± 82.77 mg L⁻¹), nitrates (2.89 ± 4.80 mg L⁻¹), ammonium (2.19 ± 1.36 mg L⁻¹) and sulphates (216.25 ± 218.54 mg L⁻¹). Ranunculus penicillatus communities are found in flowing waters with a high concentration of phosphates (0.48 ± 0.6 mg L⁻¹) and intermediate buffering capacity (683.66 ± 446.76 mg CaCO₃ L⁻¹). Both Ranunculus pseudofluitans and Ranunculus peltatus communities grow in waters with low buffering capacity (R. pseudofluitans, 385.91 ± 209.2 mg CaCO₃ L⁻¹; R. peltatus, 263.3 ± 180.36 mg CaCO₃ L⁻¹), and a low concentration of cations (R. pseudofluitans: Ca²⁺, 12.57 ± 9.42 mg L⁻¹; Mg²⁺, 3.42 ± 1.67 mg L⁻¹; R. peltatus: Ca²⁺, 15 ± 18.26 mg L⁻¹; Mg²⁺, 6.26 ± 8.89 mg L⁻¹) and nutrients (R. pseudofluitans: nitrates, 0.23 ± 0.2 mg L⁻¹; phosphates, 0.09 ± 0.1 mg L⁻¹; R. peltatus: nitrates, 0.19 ± 0.21 mg L⁻¹; phosphates, 0.09 ± 0.12 mg L⁻¹); the first in flowing waters, the latter in still waters.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: Analytical ultracentrifugation reexamination

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by subunits containing heme groups with molecular masses (M) in the range of 15 to 19 kDa, monomers of 16 kDa (d), and trimers of 51 to 52 kDa (abc) linked by nonheme structures named linkers of 24 to 32 kDa (L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12(abcd)₃L₃ (Vinogradov model) plus 144 heme groups, a value of M for HbGp oligomer of 3560 kDa can be predicted. This value is nearly 500 kDa higher than the unique HbGp M value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s⁰_20,w values of 58.1 ± 0.2 S and 59.6 ± 0.2 S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600 ± 100 and 3700 ± 100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V_bar, for HbGp was performed based on density measurements, providing a value of 0.764 ± 0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

Determination and pharmacokinetics of gastrodin in human plasma by HPLC coupled with photodiode array detector

In present study, an HPLC method coupled with photodiode array detector (HPLC-PDA) was established for determination and pharmacokinetics of gastrodin (GAS) in human plasma after an oral administration of GAS capsule. In the method, ethanol and dichloromethane were respectively used for deproteinization and purification during the sample preparation procedure. Separation of GAS was achieved on an AichromBond-AQ C18 column (5 μm, 150 mm × 4.6 mm) with the mobile phase of methanol–0.1% phosphoric acid solution (2:98, v/v) at a flow rate of 0.8 ml/min. The wavelength was set at 220 nm and the injection volume was 20 μl. Under the conditions, the calibration curve was linear within the concentration range of 50–4000 ng/ml with the correlation coefficient (r) of 0.99554 (weight = 1/X²) and the lower limit of quantification (LLOQ) was 50 ng/ml. The inter- and intra-day precisions were less than 11% and the accuracies (%) were within the range of 95.55–103.78%. The extraction recoveries were over 65% with RSDs less than 5.50%. The GAS was proved to be stable under tested conditions. Thus, the method was valid enough to be applied for pharmacokinetic study of GAS in human plasma. The pharmacokinetic parameters of GAS in human plasma after an oral administration of 200 mg GAS capsule were described as: C_max, 1484.55 ± 285.05 ng/ml; T_max, 0.81 ± 0.16 h; t_1/2α, 3.78 ± 2.33 h; t_1/2β, 6.06 ± 3.20 h; t_1/2Ka, 0.18 ± 0.53 h; K₁₂, 0.18 ± 0.41/h; K₂₁, 0.20 ± 0.16/h; K₁₀, 4.11 ± 15.81/h; V1/F, 180.35 ± 89.44 L; CL/F, 62.50 ± 140.03 l/h; AUC_0→t, 5619.41 ± 1972.88 (ng/ml) h; and AUC_0→∞, 7210.26 ± 3472.74 (ng/ml) h, respectively. These will be useful for the clinical application of GAS.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

Magnitude of sarcomere extension correlates with initial sarcomere length during lengthening of activated single fibers from soleus muscle of rats

A laser-diffraction technique was developed that rapidly reports the lengths of sarcomeres (L_s) in serially connected sectors of permeabilized single fibers. The apparatus translates a laser beam along the entire length of a fiber segment within 2 ms, with brief stops at each of 20 contiguous sectors. We tested the hypothesis that during lengthening contractions, when maximally activated fibers are stretched, sectors that contain the longer sarcomeres undergo greater increases in L_s than those containing shorter sarcomeres. Fibers (n = 16) were obtained from the soleus muscles of adult male rats and the middle portions (length = 1.05 ± 0.11 mm; mean ± SD) were investigated. Single stretches of strain 27% and a strain rate of 54% s⁻¹ were initiated at maximum isometric stress and resulted in a 19 ± 9% loss in isometric stress. The data on L_s revealed that 1), the stretch was not distributed uniformly among the sectors, and 2), during the stretch, sectors at long L_s before the stretch elongated more than those at short lengths. The findings support the hypothesis that during stretches of maximally activated skeletal muscles, sarcomeres at longer lengths are more susceptible to damage by excessive strain.     

Complexation of curium(III) by adenosine 5′-triphosphate (ATP): A time-resolved laser-induced fluorescence spectroscopy (TRLFS) study

The complex formation of curium(III) with adenosine 5′-triphosphate (ATP) was determined by time-resolved laser-induced fluorescence spectroscopy (TRLFS). The interaction between soluble species of curium(III) with ATP was studied at trace Cm(III) concentrations (3 × 10⁻⁷ M). The concentrations of ATP were varied between 6.0 × 10⁻⁷ and 1.5 × 10⁻⁴ M in the pH range of 1.5-7.0 using 0.154 M NaCl as background electrolyte.Three Cm-ATP species, M_pH_qL_r, could be identified from the fluorescence emission spectra: (i) CmH₂ATP⁺ with a peak maximum at 598.6 nm, (ii) CmHATP with a peak maximum at 600.3 nm, and (iii) CmATP⁻ with a peak maximum at 601.0 nm. The formation constants of these complexes were calculated from TRLFS measurements to be log β₁₂₁ = 16.86 ± 0.09, log β₁₁₁ = 13.23 ± 0.10, and log β₁₀₁ = 8.19 ± 0.16. The hydrated Cm-ATP species showed fluorescence lifetimes between 88 and 96 μs; whereas the CmATP⁻ complex has a significantly longer fluorescence lifetime of 187 ± 7 μs.     

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4 °C, 12 °C, 21 °C, and 37 °C, the activation energy for water transport was determined to be E_a = 51 ± 3 kJ/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130 °C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below −15 °C.     

Complex formation between protactinium(V) and sulfate ions at 10 and 60 °C

Protactinium complexation with sulfate ions was studied with the element at tracer scale (C_Pa ∼ 10⁻¹² M) by solvent extraction method. The involved aqueous system was Pa(V)/H₂O/HClO₄/Na₂SO₄/NaClO₄ at 10 and 60 °C. The extraction experiments were conducted using the chelating agent thenoyltrifluoroacetone (TTA) in toluene. For both values of temperature, a systematic study was performed in order to determine the formation constants (β₁, β₂ and β₃) of sulfate complexes of Pa(V) at different ionic strength. For each temperature, the extrapolation of these constants to zero ionic strength was performed using the Specific Interaction Theory, leading to values of 2.8 ± 0.5, 6.5 ± 0.5, 7.8 ± 0.5 at 10 °C and 4.3 ± 0.3, 8.4 ± 1.3, 9.6 ± 0.4 at 60 °C. Interaction coefficients involving the sulfate complexes of protactinium(V) were also derived.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

(E)-2-(2-(2-hydroxyphenyl)hydrazono)-1-phenylbutane-1,3-dione: Tautomery and coordination to copper(II)

(E)-2-(2-(2-hydroxyphenyl)hydrazono)-1-phenylbutane-1,3-dione (H₂L) was synthesized by azocoupling of diazonium salt of 2-hydroxyaniline with 1-phenylbutane-1,3-dione and characterized by IR, ¹H and ¹³C NMR spectroscopies and X-ray diffraction analysis. In solution, H₂L exists as a mixture of the enol-azo and hydrazone tautomeric forms and a decrease of temperature and of solvent polarity shifts the tautomeric balance to the hydrazone form. In the solid state, H₂L crystallizes from ethanol-water in the monohydrate hydrazone form, as shown by X-ray analysis. The dissociation constants of H₂L (pK₁ = 5.98 ± 0.04, pK₂ = 9.72 ± 0.03) and the stability constants of its copper(II) complex (log β₁ = 11.01 ± 0.07, log β₂ = 20.19 ± 0.08) were determined by the potentiometric method in aqueous-ethanol solution. The copper(II) complex [Cu₂(μ-L)₂]_n was isolated in the solid state and found by X-rays to be a coordination polymer of a binuclear core with a distorted square pyramidal metal coordination geometry.     

Comparative pharmacokinetics of baicalin and wogonoside by liquid chromatography–mass spectrometry after oral administration of Xiaochaihu Tang and Radix scutellariae extract to rats

The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC–MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C₁₈ column (150 mm × 4.6 mm, i.d.: 5 μm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/z 447 for baicalin, m/z 461 for wogonoside and m/z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156 μg mL⁻¹ for baicalin and 0.08832 to 4.416 μg mL⁻¹ for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155 μg mL⁻¹ for baicalin and 0.04416 μg mL⁻¹ for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t_1/2 of baicalin was 3.60 ± 0.90 and 5.64 ± 1.67, the C_max1 was 1.64 ± 0.99 and 5.66 ± 2.02, the t_max1 was 0.13 ± 0.05 and 0.20 ± 0.07, the C_max2 was 2.43 ± 0.46 and 3.18 ± 1.66, and the t_max2 were 6.40 ± 1.67 and 5.66 ± 2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t_1/2 of wogonoside was 4.97 ± 1.68 and 7.71 ± 1.55, the C_max1 was 1.39 ± 0.83 and 1.45 ± 0.37, the t_max1 was 0.21 ± 0.20 and 0.17 ± 0.01, the C_max2 was 1.90 ± 0.55 and 1.42 ± 0.70, and the t_max2 was 5.60 ± 1.67 and 5.20 ± 1.79, respectively. A significant difference (p < 0.05) was observed for t_1/2, and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased.     

Fast trigonal/tetragonal interconversion followed by slow chelate-ring closure in the complexation of boronic acids

Which of the step is rate determining in the complexation of boronic acid with bidentate ligands, the first trigonal/tetragonal interconversion step or the following chelate-ring closure step ? No one could have ever answered this simple question clearly. In this study, we for the first time gave an explicit answer to this question. Reaction systems were devised for kinetic measurements to answer this simple question. At first, pK_a values of 15 boronic acids were determined spectrophotometrically at I = 1.0 M and 25 °C: pK_a = 6.40 ± 0.06 for 3,5-Cl₂PhB(OH)₂, 6.62 ± 0.03 for 3,5-(CF₃)₂PhB(OH)₂, 6.95 ± 0.09 for 3,5-Br₂PhB(OH)₂, 7.04 ± 0.03 for 3-NO₂PhB(OH)₂, 7.08 ± 0.03 for 3,5-F₂PhB(OH)₂, 7.12 ± 0.02 for 2,4-F₂PhB(OH)₂, 7.39 ± 0.02 for 4-CF₃PhB(OH)₂, 7.50 ± 0.02 for 3-FPhB(OH)₂, 7.83 ± 0.02 for 2-FPhB(OH)₂, 7.87 ± 0.01 for 3-CF₃PhB(OH)₂, 8.11 ± 0.01 for 2-ClPhB(OH)₂, 8.14 ± 0.03 for 3-(COOH)PhB(OH)₂, 8.15 ± 0.03 for 3-(CH₃CO)Ph(OH)₂, 8.20 ± 0.01 for 3-ClPhB(OH)₂, and 8.66 ± 0.05 for 4-FPhB(OH)₂. The kinetic results for the reactions of some of these boronic acids with 4-isopropyltropolone clearly show that the rate-determining step is not the interconversion step but the chelate-ring closure step.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.