Incubation In Solution Research Articles

Effect of the β-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases

Glucuronidation studies using microsomes and recombinant uridine diphosphoglucuronosyltransferases (UGTs) can be complicated by the presence of endogenous beta-glucuronidases, leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used beta-glucuronidase inhibitor, although it is not clear whether this reagent should be added routinely to glucuronidation incubations. Here we have determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and recombinant UGTs (rUGTs). Despite the use of buffered incubation solutions, it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM did not enhance any of the glucuronidation activities evaluated that could be considered consistent with inhibition of beta-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for glucuronidation of 5-hydroxytryptamine and estradiol by pHLMs, with a 35% decrease at 20 mM saccharolactone concentration. Endogenous beta-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes and insect-cell expressed rUGTs, but not for kidney, intestinal or human embryonic kidney HEK293 microsomes. However, the extent of hydrolysis was relatively small, representing only 9-19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations. If saccharolactone is used, concentrations should be titrated to achieve activity enhancement without inhibition.

LC/MS/MS for identification of in vivo and in vitro metabolites of jatrorrhizine

The in vivo and in vitro metabolism of jatrorrhizine has been investigated using a specific and sensitive LC/MS/MS method. In vivo samples including rat feces, urine and plasma collected separately after dosing healthy rats with jatrorrhizine (34 mg/kg) orally, along with in vitro samples prepared by incubating jatrorrhizine with rat intestinal flora and liver microsome, respectively, were purified using a C(18) solid-phase extraction cartridge. The purified samples were then separated with a reversed-phase C(18) column with methanol-formic acid aqueous solution (70:30, v/v, pH3.5) as mobile phase and detected by on-line MS/MS. The structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, seven new metabolites were found in rat urine, 13 metabolites were detected in rat feces, 11 metabolites were detected in rat plasma, 17 metabolites were identified in intestinal flora incubation solution and nine metabolites were detected in liver microsome incubation solution. The main biotransformation reactions of jatrorrhizine were the hydroxylation reaction, the methylation reaction, the demethylation reaction and the dehydrogenation reaction of parent drug and its relative metabolites. All the results were reported for the first time, except for some of the metabolites in rat urine.

Sorptive interaction between goethite and strongly reducing organic substances from anaerobic decomposition of green manures

Strongly reducing organic substances (SROS) and iron oxides exist widely in soils and sediments and have been implicated in many soil and sediment processes. In the present work, the sorptive interaction between goethite and SROS derived from anaerobic decomposition of green manures was investigated by differential pulse voltammetry (DPV). Both green manures, Astragalus sinicus ( Astragalus) and Vicia varia ( Vicia) were chosen to be anaerobically decomposed by the mixed microorganisms isolated from paddy soils for 30 d to prepare different SROS. Goethite used in experiments was synthesized in laboratory. The anaerobic incubation solutions from green manures at different incubation time were arranged to react with goethite, in which SROS concentration and Fe(II) species were analyzed. The anaerobic decomposition of Astragalus generally produced SROS more in amount but weaker in reducibility than that of Vicia in the same incubation time. The available SROS from Astragalus that could interact with goethite was 0.69 ± 0.04, 0.84 ± 0.04 and 1.09 ± 0.03 cmol kg −1 as incubated for 10, 15 and 30 d, respectively, for Vicia, it was 0.12 ± 0.03, 0.46 ± 0.02 and 0.70 ± 0.02 cmol kg −1. One of the fates of SROS as they interacted with goethite was oxidation. The amounts of oxidizable SROS from Astragalus decreased over increasing incubation time from 0.51 ± 0.05 cmol kg −1 at day 10 to 0.39 ± 0.04 cmol kg −1 at day 30, but for Vicia, it increased with the highest reaching to 0.58 ± 0.04 cmol kg −1 at day 30. Another fate of these substances was sorption by goethite. The SROS from Astragalus were sorbed more readily than those from Vicia, and closely depended upon the incubation time, whereas for those from Vicia, the corresponding values were remarkably less and apparently unchangeable with incubation time. The extent of goethite dissolution induced by the anaerobic solution from Vicia was greater than that from Astragalus, showing its higher reactivity.

An evaluation of methods for quantifying the enzymatic degradation of red seaweed Grateloupia turuturu

For better exploitation of the red seaweed Grateloupia, enzymatic digestion of the thallus may be a way to increase access to metabolites of industrial interest. With this aim, we have tried to find a method to quantify the efficiency of enzymatic digestion. Vegetative algal material was treated with polysaccharidases (Onozuka R-10 cellulase, agarase, and Ultraflo L mixture). The proportion of degraded surface area was determined by microscopic measurement of the residue surface using imaging software and compared with the analysis of carbohydrates and R-phycoerythrin released in the incubation solution. Both the reducing carbohydrate concentration and percentage of degraded surface area appeared the most reliable methods to study enzymatic efficiency. The amount of solubilized total carbohydrates, and particularly that of R-phycoerythrin, showed non-specific variations, so no conclusions could be drawn. The application of this procedure to the screening of the efficient digestion of Grateloupia material demonstrated that cell walls were only partially digested by polysaccharidase enzymes alone. The Ultraflo L mixture and Onozuka R-10 cellulase produced a greater degradation of Grateloupia tissues and a higher release of reducing carbohydrates, whereas agarase did not display any specific action. Thus, the proposed procedure based on the quantification of residue surface area seems to be an accurate method to analyze enzymatic digestion. Other tests using different concentrations and combinations of enzymes are now required.

Enzymatic removal of plant residues from wool: Application of experimental design techniques for optimization parameters

This study was undertaken to find the optimum conditions of a new enzymatic process to remove plant residues from wool. Commercial enzymatic preparations of Celluclast 1.5 L and Pectinex Ultra SP-L were selected in order to hydrolyze the polysaccharides in primary plant cell walls and middle lamella, resulting into more fragile residues easier to be removed. Since it was intended to define the optimal conditions for enzyme application, a four-factor central composite design was selected to study the effects of pH, temperature, enzyme concentration and wetting agent concentration, on the two selected responses, i.e., soluble reducing sugars (RS) and alkali solubility (AS) of wool to detect plant degradation and to evaluate wool quality, respectively. Results demonstrated that enzyme concentration was the most significant effect in plant residues degradation. A total enzyme concentration loading of 20 mL of both diluted enzymatic preparations in equal parts per 1 L of incubation solution (42.970 U/L of Celluclast preparation and PG 29.3 nkat/L + PME 2.537 nkat/L of Pectinex preparation), yielded an equivalent amount of 240.127 mg of glucose per 1.0 g of plant residue, at the optimal conditions: 40.56 °C, pH 4.0 and 1 mL Plurafac/L. SEM analysis has indicated an identical and important degradation of the plant residues, when compared to the conventional carbonization process, and wool quality has been preserved.

In Situ Mineralization of Hydroxyapatite on Electrospun Chitosan‐Based Nanofibrous Scaffolds

A biocomposite of hydroxyapatite (HAp) with electrospun nanofibrous scaffolds was prepared by using chitosan/polyvinyl alcohol (CS/PVA) and N-carboxyethyl chitosan/PVA (CECS/PVA) electrospun membranes as organic matrix, and HAp was formed in supersaturated CaCl2 and KH2PO4 solution. The influences of carboxylic acid groups in CECS/PVA fibrous scaffold and polyanionic additive poly(acrylic acid) (PAA) in the incubation solution on the crystal distribution of the HAp were investigated. Field-emission scanning electron microscopy (FE-SEM), energy-dispersive spectroscopy (EDS), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared (FTIR) were used to characterize the morphology and structure of the deposited mineral phase on the scaffolds. It was found that addition of PAA to the mineral solution and use of matrix with carboxylic acid groups promoted mineral growth and distribution of HAp. MTT testing and SEM imaging from mouse fibroblast (L929) cell culture revealed the attachment and growth of mouse fibroblast on the surface of biocomposite scaffold, and that the cell morphology and viability were satisfactory for the composite to be used in bioapplications.

Surface Plasmon Resonance Analysis of Alzheimer's β-Amyloid Aggregation on a Solid Surface: From Monomers to Fully-Grown Fibrils

We analyzed the aggregation of Alzheimer's beta-amyloid (1-42) (Abeta42) peptides from fresh monomers to fully grown fibrils by using in situ surface plasmon resonance (SPR) spectrometry and ex situ atomic force microscopy (AFM). To immobilize Abeta42 peptide on an SPR chip surface, different carboxy-terminated surfaces were investigated: (1) self-assembled monolayer of 11-mercaptoundecanoic acid and (2) carboxylated dextran-modified surface. It was found that the carboxylated dextran surface was more appropriate due to a much lower degree of nonspecific binding. By using the carboxylated dextran surface, we further investigated effects of key environmental factors, such as the density of surface-bound Abeta42, the concentration of Abeta42 in solution phase, and the presence of Fe3+ ions on Abeta42 fibrillation. The increase in either the surface density of Abeta42 or its concentration in incubation solution highly accelerated the formation of amyloid fibrils on the chip surface. The presence of Fe3+ ions in the incubation solution induced significantly denser aggregates, resulting in a nearly 6-fold increase of SPR angle shift. This work shows that SPR analysis coupled with AFM can be effectively used for analyzing amyloid aggregation and deposition on a solid surface from the very beginning to fully grown fibrils.

Modification of Nafion membrane using interfacial polymerization for vanadium redox flow battery applications

In order to reduce the permeation of vanadium ions across the ion exchange membrane during the operation of vanadium redox flow battery (VRB) based on Nafion membrane, the interfacial polymerization was applied to form a cationic charged layer on the surface of Nafion 117 membrane. The area resistance and the permeability of vanadium ions were measured. The results indicate that comparing with the unmodified Nafion membrane, the modification of Nafion membrane results in a dramatic reduction in crossover of vanadium ions across the membrane and a little higher area resistance of the membrane. As a result, the columbic efficiency for the VRB single cell based on the modified Nafion membrane(VRB-modified Nafion), which is related to the concentration of the incubation solution of polyethylenimine (PEI), was increased significantly. The value is 96.2–97.3%, which is higher than that obtained with the VRB single cell based on unmodified Nafion membrane(VRB-Nafion) (around 93.8%). Due to the little higher area resistance caused by the modification, the voltage efficiency of VRB-modified Nafion is lower than that of VRB-Nafion. Furthermore, the water transfer across the modified membrane was also reduced. The ion exchange capacity (IEC) of the modified Nafion membrane was also evaluated. The formation of the thin cationic charged layer on the membrane surface was confirmed by IR spectra analysis.

Biosorption of metal and salt tolerant microbial isolates from a former uranium mining area. Their impact on changes in rare earth element patterns in acid mine drainage

The concentration of metals in microbial habitats influenced by mining operations can reach enormous values. Worldwide, much emphasis is placed on the research of resistance and biosorptive capacities of microorganisms suitable for bioremediation purposes. Using a collection of isolates from a former uranium mining area in Eastern Thuringia, Germany, this study presents three Gram-positive bacterial strains with distinct metal tolerances. These strains were identified as members of the genera Bacillus, Micrococcus and Streptomyces. Acid mine drainage (AMD) originating from the same mining area is characterized by high metal concentrations of a broad range of elements and a very low pH. AMD was analyzed and used as incubation solution. The sorption of rare earth elements (REE), aluminum, cobalt, copper, manganese, nickel, strontium, and uranium through selected strains was studied during a time course of four weeks. Biosorption was investigated after one hour, one week and four weeks by analyzing the concentrations of metals in supernatant and biomass. Additionally, dead biomass was investigated after four weeks of incubation. The maximum of metal removal was reached after one week. Up to 80% of both Al and Cu, and more than 60% of U was shown to be removed from the solution. High concentrations of metals could be bound to the biomass, as for example 2.2 mg/g U. The strains could survive four weeks of incubation. Distinct and different patterns of rare earth elements of the inoculated and non-inoculated AMD water were observed. Changes in REE patterns hint at different binding types of heavy metals regarding incubation time and metabolic activity of the cells.

Characteristics of amino acid uptake in barley

Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten kinetics. The affinity constant (K m) was in the range 19.6–33.2 μM. These K m values are comparable to reported values for soil micro-organisms.

Decomposition of aqueous organic compounds in the Atacama Desert and in Martian soils

Carbon‐13 labeled formate, alanine, and glucose decompose when added in aqueous solution to soils collected from the “Mars‐like” Yungay region (S 24° 4′ 9.6″, W 69° 51′) of the Atacama Desert. During the first 5 d of incubation, alanine (5 mM) and glucose (5 mM) solutions decomposed at rates of 0.1 to 0.2 μmol/d, and formate solution (50 mM) decomposed at rates of 0.4 to 1.6 μmol/d. The observation of approximately equal 13CO2 initial production rates by soils treated with D‐glucose and L‐alanine, compared to soils treated with L‐glucose and D‐alanine, indicates the presence of one or more nonbiological chemical decomposition mechanisms. An increase in the decomposition rates of D‐glucose and L‐alanine, compared to L‐glucose and D‐alanine ∼5 d after the addition of these organics, demonstrates that the soils are also biologically active. When treated with sodium formate solution, tested soils released 13CO2 gas in a manner that reproduces the initial gas release observed in the Mars Viking Labeled Release (LR) experiment. Our results indicate that the 13CO2 produced in Yungay soils is consistent with an initial phase of nonbiological decomposition followed by biological decomposition of added organics. Heat treatment of Yungay soils eliminated all CO2 production, while in the Viking LR experiment, the initial rapid CO2 release was eliminated by heat treatment, but a slower secondary CO2 production was not. Our results indicate that the mechanism for the decomposition of organics in Yungay soils is different from the processes observed in the Viking LR experiment.

Peculiarities of functioning of wheat cut-off root cells at inhibition of complexes I and II of mitochondrial respiratory chain

Combined action of rotenone and malonate, inhibitors of complexes I and II of the mitochondrial electron transport chain (ETC), on wheat cut-off root seedlings was studied after 6 h of incubation. Intensity of oxygen consumption and release of potassium ions into incubation medium were determined simultaneously with the study of changes in cell ultrastructure. Malonic acid was added 1 h after the root incubation in the rotenone solution and produced inhibition of respiration, as well as a greater release of K+ into the incubation solution as compared with effect of rotenone alone. After 2 h of the combined action of these inhibitors, many mitochondria acquired a toroidal shape, thereby increasing the outer surface. For the ensuing hours, stimulation of oxygen consumption by the roots and a decrease of K+ content in the incubation medium were observed. Mitochondria once again acquired a round or oval shape and compensation-reparation processes took place. Contacts of endoplasmic reticulum channels with mitochondria were observed, which seems to be due to the synthesis of the enzyme splitting malonate to acetyl-CoA, which in turn can be included both into the Krebs cycle and into lipogenesis. It is suggested that the toroidal form of mitochondria is associated with the activation of the external NAD(P)H-dehydrogenase of the inner mitochondrial membrane, as under these conditions, at the inhibition of the ETC complexes I and II, the activity of other dehydrogenises is blocked. Thus, the use of the external NAD(P)H allows the activity of the ETC mitochondria to be restored, which facilitates the course of the reparation processes and allows cells to be adapted to this action.

Recognition of Glycoprotein Peroxidase via Con A-Carrying Self-Assembly Layer on Gold

We have successfully fabricated a self-assembled layer of concanavalin A (Con A) on a gold surface for recognition of glycoproteins. The type IV Con A is covalently bound to 11-mercaptoundecanoic acid (MUA) on gold with a 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) linkage. The binding interaction between glycoproteins and self-assembled Con A is studied using horseradish peroxidase (HRP) as a model glycoprotein. Voltammetric, electrochemical impedance studies, and photometric activity measurements show the presence of both specific and nonspecific bindings of HRP to the Con A interface. The specific binding is attributed to the Con A-sugar interaction where Con A selectively recognizes the glycosylation sites of HRP. The catalytic current of the HRP-loaded electrode, because of catalytic oxidation of thionine in the presence of hydrogen peroxide (H2O2), is found to be proportional to the HRP concentrations in the incubation solution. A linear correlation coefficient of 0.993 was obtained over a wide HRP concentration range of 12.5 microg/mL to 1 mg/mL. The approach described in this study provides a simple yet selective means to immobilize glycoproteins on a solid support. The specific binding achieved is desirable in biosensor fabrication, glycoprotein separation, recognition, and purification as well as in drug-releasing systems.

Characterization of in vivo and in vitro Metabolic Pathway of Anisodamine by Liquid Chromatography‐Tandem Mass Spectrometry

In vivo and in vitro metabolisms of anisodamine were investigated using a highly specific and sensitive LC‐MSn method. Feces, urine, and plasma samples were collected individually after ingestion of 25 mg/kg anisodamine to healthy rats. Rat feces and urine samples were cleaned up by a liquid‐liquid extraction and a solid phase extraction procedure (C18 cartridges), respectively. Methanol was added to rat plasma samples to precipitate plasma proteins. Anisodamine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively. The metabolites in the incubation solution were extracted with ethyl acetate. Then, these pretreated in vivo and in vitro samples, were injected into a reversed‐phase C18 column with mobile phase of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by an on‐line MSn system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (ΔM), retention times and full scan MSn spectra with those of the parent drug. Fifteen new metabolites (aponoranisodamine, apoanisodamine, methoxyanisodamine, hydroxy‐methoxyanisodamine, trihydroxyanisodamine, dimethoxyanisodamine, dihydroxy‐methoxyanisodamine, tetrahydroxyanisodamine, hydroxy‐dimethoxyanisodamine, trihydroxy‐methoxyanisodamine, dihydroxy‐dimethoxyanisodamine, tetrahydroxy‐methoxyanisodamine, trihydroxy‐dimethoxyanisodamine, dihydroxy‐trimethoxyanisodamine, and hydroxy‐tetramethoxyanisodamine) were identified in rat urine after ingesting anisodamine. Seven metabolites (nor‐6β‐hydroxytropine, 6β‐hydroxytropine, tropic acid, aponoranisodamine, apoanisodamine, noranisodamine, and anisodamine N‐oxide) and the parent drug were detected in rat feces. Six metabolites (nor‐6β‐hydroxytropine, 6β‐hydroxytropine, tropic acid, apoanisodamine, hydroxyanisodamine, and anisodamine N‐oxide) and the parent drug are detected in rat plasma. Only apoanisodamine was detected in the homogenized liver incubation mixture. The hydrolyzed metabolites (6β‐hydroxytropine and tropic acid) and the dehydrated metabolite of anisodamine were found in the rat intestinal flora incubation mixture.

An amperometric immunosensor for separation-free immunoassay of CA125 based on its covalent immobilization coupled with thionine on carbon nanofiber

A carbon nanomaterial, soluble carbon nanofiber, was used for the first time to construct an immunosensor for a rapid separation-free immunoassay. The acidic oxidation of the carbon nanofiber provided its solubility and wettability for convenient preparation of a porous carbon nanofiber membrane and a larger number of active sites for covalent binding of carcinoma antigen-125 (CA125) and thionine as electron transfer mediator. This matrix was a suitable environment for the immobilized protein. The immobilized HRP-labeled immunoconjugate showed good enzymatic activity for the oxidation of thionine by hydrogen peroxide. With a competitive mechanism, the differential pulse voltammetric peak current of this system decreased linearly with increasing CA125 concentration (from 2 to 75 U/ml) in the incubation solution. The CA125 immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility with a detection limit of 1.8 U/ml. The soluble carbon nanofiber is a novel method for preparation of immunosensors.

Distinct Iron Binding Property of Two Putative Iron Donors for the Iron-Sulfur Cluster Assembly: IscA AND THE BACTERIAL FRATAXIN ORTHOLOG CyaY UNDER PHYSIOLOGICAL AND OXIDATIVE STRESS CONDITIONS

Frataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the iron-sulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions.

LC–MS for Identification and Elucidation of the Structure of In-Vivo and In-Vitro Metabolites of Atropine

In-vivo and in-vitro metabolism of atropine has been investigated by use of a highly specific and sensitive LC–MSn method. Feces, urine, and plasma samples were collected separately after ingestion of 25 mg kg−1 atropine by healthy rats. Rat feces and urine samples were cleaned by liquid–liquid extraction and by solid-phase extraction (on C18 cartridges), respectively. Methanol was added to rat plasma samples to precipitate plasma proteins. Atropine was incubated, in vitro, with homogenized liver and with intestinal flora from rats. The metabolites in the incubation solution were extracted with ethyl acetate. These pretreated samples were then analyzed by reversed-phase high-performance liquid chromatography on a C18 column with methanol–ammonium acetate (2 mm, adjusted to pH 3.5 with formic acid), 70:30 (v/v), as mobile phase. Detection was by on-line MSn. Identification and elucidation of the structure of the metabolites were achieved by comparing molecular mass (ΔM), retention-times, and full-scan MSn spectra with those of the parent drug. Ten new metabolites (aponoratropine, apoatropine, hydroxymethoxyatropine, trihydroxyatropine, dimethoxyatropine, dihydroxymethoxyatropine, hydroxydimethoxyatropine, trihydroxymethoxyatropine, dihydroxydimethoxyatropine, and tropic acid) were identified in rat urine after ingestion of atropine. Nine metabolites (nortropine, tropine, aponoratropine, apoatropine, noratropine, hydroxyatropine, hydroxyatropine N-oxide, hydroxymethoxyatropine, and tropic acid) and the parent drug were detected in rat feces. Five metabolites (nortropine, tropine, tropic acid, apoatropine, and hydroxyatropine) and the parent drug were detected in rat plasma. Only two metabolites (apoatropine and noratropine) were detected in the homogenized liver incubation mixture. The hydrolyzed metabolites (tropine and tropic acid) and dehydrated metabolite apoatropine were found in the rat intestinal flora incubation mixture.

Rapid breakdown of exogenous extracellular hydrogen peroxide by lichens

All organisms, even highly stress‐tolerant lichens, produce a variety of reactive oxygen species (ROS) during and after stress. Furthermore, the cell walls of some lichens in Suborder Peltigerineae contain laccases, and therefore can produce quinone radicals that can break down to yield ROS. While the extracellular ROS produced by these enzymes probably play important roles in the biology of these lichens, they may also be potentially harmful and need to be rapidly broken down. To test this, rates of breakdown of exogenously supplied H2O2 were measured in a range of lichen species. Considerable diversity existed in rates of H2O2 breakdown but rates were on average almost double in members of Suborder Peltigerineae. While all lichens tested appeared to lack extracellular peroxidases and catalases, enzymes normally involved in breaking down H2O2, extracellular tyrosinase activity could be readily detected in the Peltigerineae. A role for tyrosinases in H2O2 breakdown was supported by the results from experiments involving inhibitors, and demonstration of the simultaneous release into an incubation solution of tyrosinase activity and the ability to breakdown H2O2. Rates of breakdown were very high, and tyrosinase appeared to break down H2O2 by a catalase‐like mechanism. However, significant rates of breakdown of H2O2 also occurred in species that did not possess cell wall redox enzymes. These species probably took up the exogenously supplied H2O2 intracellularly and then broke it down by the usual catalases and peroxidases. The importance of H2O2 degradation is discussed in terms of its possible role in defence against the harmful effects of ROS.

Inhibitory effect of osmotic concentration, potassium and pH on motility of the sperm of the North American burbot Lota lota maculosa

Seminal plasma factors maintaining North American (NA) burbot Lota lota maculosa sperm quiescent were examined. Sperm were diluted into buffered saline solutions of various compositions and motility assessed. After 1 h in these solutions at 10° C, aliquots of the suspension were diluted with tap water and motility again assessed. Dilution of sperm in an incubation solution containing Ca2+ in the absence of K+ initiated sperm motility resulting in low motility when sperm were subsequently diluted in tap water. Incubation solutions with osmolalities >200 mOsm kg−1 and containing 12·5 mM K+ prevented the onset of sperm motility and were associated with maximal sperm motility upon dilution in tap water. Sperm maintained at lower osmolalities exhibited limited motility upon dilution in tap water indicating interdependence between K+ and osmolality in maintaining sperm quiescent in the presence of Ca2+. Sperm kept in incubation solution at pH values < c. 7·5 for 1 h demonstrated reduced motility when subsequently diluted in tap water. That motility of sperm was pH sensitive was further indicated by CO2 inhibition of motility. Therefore, NA burbot sperm are probably maintained in an immotile state, yet with potential for motility, by combination of high K+, osmolality and possibly pH. The results from this study differ from published information on sperm quiescence in the temporally and geographically distinct Eurasian burbot Lota lota lota.

Gap junction hemichannel-mediated release of glutathione from cultured rat astrocytes

Release of glutathione (GSH) from astrocytes is essential for the supply of neurons with the GSH precursor cysteine. In order to test whether gap junction hemichannels could contribute to GSH release from astrocytes, we incubated astrocyte-rich primary cultures from neonatal rat brain in the absence of divalent cations, a condition that is known to increase the opening probability of hemichannels. During incubation in divalent cation free incubation solution (DCFS) the cells remained viable and released about 50% of the initial cellular GSH within 15 min. This extracellular GSH accumulation in DCFS was lowered by the presence of Ca 2+ in a concentration dependent manner with a half-maximal inhibition at a Ca 2+ concentration of 107 ± 46 μM. Extracellular GSH accumulation in DCFS was also blocked by the divalent cations Mg 2+, Ba 2+ and Sr 2+ as well as by the known gap junction inhibitors carbenoxolone (CBX), flufenamic acid (FFA) and lanthanum chloride. In contrast, the P2X 7 receptor blocker brilliant blue G (BBG) did not affect GSH release in divalent cation free solution. This pharmacological profile strongly suggests that astrocytes are able to release GSH via open hemichannels. This release of GSH may have severe consequences for the antioxidative defense and for the GSH homeostasis in pathological brain.