In situ visualization of glucocerebrosidase in human skin tissue: zymography versus activity-based probe labeling.

Epidermal ß-glucocerebrosidase (GBA1), an acid ß-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: i) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-ß-D-glucopyranoside or resorufin-ß-D-glucopyranoside; and ii) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro-ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various ß-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio.

A novel live cell assay to measure diacylglycerol lipase α activity.

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

Lactobacillus gasseri SBT2055 suppresses fatty acid release through enlargement of fat emulsion size in vitro and promotes fecal fat excretion in healthy Japanese subjects.

BACKGROUND: Lactobacillus gasseri SBT2055 (LG2055) has been shown to prevent abdominal adiposity, and suppression of lipid absorption is considered a possible mechanism, detail of which, however, are poorly understood. In the present study, we evaluated the effects of LG2055 on fat hydrolysis by determining pancreatic lipase activity and fat emulsion properties in vitro. We also examined whether LG2055 influences fecal fat excretion in humans. METHODS: Pancreatic lipase activity was investigated in vitro using an artificially prepared fat emulsion and 4-methylumbelliferyl oleate (4-MUO) as substrates. The concentrations of free fatty acids and 4-methylumbelliferone were quantified. Fat emulsion droplet size was measured using a particle size analyzer. The clinical study was performed as a double-blind, randomized, placebo-controlled trial. Subjects consumed 100 g of fermented milk (FM)/d, either with or without LG2055 supplementation, for seven days. Fecal samples were collected during three-day pre-observational and FM intake periods and fecal fat levels were determined. RESULTS: LG2055 dose-dependently suppressed lipase activity in the fat emulsion assay but not in the 4-MUO assay. LG2055 dose-dependently increased fat emulsion droplet size. The effects of LG2055 on lipase activity and fat emulsion properties were increased compared with four other tested strains (Lactobacillus gasseri SBT0317, Lactobacillus gasseri JCM1131T, Lactobacillus. delbrueckii subsp. bulgaricus JCM1002T and Streptococcus thermophilus ATCC19258T). In our clinical study, fecal fat level after FM intake was significantly increased compared with that observed before FM intake in the LG2055-containing active FM group but not the control FM group lacking LG2055. CONCLUSIONS: LG2055 increased fat emulsion droplet size, resulting in the suppression of lipase-mediated fat hydrolysis. The influence of LG2055 on the physicochemical properties of fat emulsion provides a mechanism for the probiotic-mediated suppression of lipid absorption and promotion of fecal fat excretion in humans. TRIAL REGISTRATION: UMIN000015772.

Comparative biological study of roots, stems, leaves, and seeds of Angelica shikokiana Makino.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica shikokiana has been used as a health food for its anticancer, anti-inflammatory, antibacterial, antiallergic, and blood vessel dilating effects in Japan. It can also be used to prevent and treat hepatitis, diabetes, hyperlipidemia, and arteriosclerosis. AIM OF THE STUDY: The present study was designed to compare the biological activities such as melanin synthesis inhibitory, anti-allergy, anti-lipase, anti-bacterial, anti-oxidant, and neuroprotective activities of different parts of the plant that may justify the use of this plant in folk medicine. MATERIAL AND METHODS: The roots, stems, leaves and, seeds of Angelica shikokiana were separately extracted with water and ethanol. Each extract was examined for melanin synthesis inhibitory and anti-allergy activity on B16-melanoma and RBL-2H3 cells using IgE and A23187 as a stimulant for ß-hexosaminidase release, respectively. We also evaluated the inhibition of two enzymes, lipase and acetylcholine esterase, and of the bacterial growth of two species, Escherichia coli and Staphylococcus aureaus. The anti-oxidant activity was determined using oxygen radical anti-oxidant capacity, ORAC assay and its relation to the phenolic content was estimated using the Folin-Ciocalteu method. Besides, the protective effect of the extracts against H2O2-induced oxidative stress in mouse neuroblastoma, Neuro-2A cells was investigated. RESULTS: The most active extract exhibiting melanin synthesis inhibition (63%) and at the same time with low cytotoxicity (15%) was the ethanol extract of roots at 20 µg/ml, followed by the ethanol extract of stems (57% inhibition, 5% cytotoxicity). On the other hand, the highest inhibitions of ß-hexosaminidase release were recorded for the ethanol extract of leaves with IC50 value of 6.89 µg/ml followed by the water extract of the seeds and leaves with IC50 value of 78.32 and 88.44 µg/ml, respectively. For anti-lipase assay, ethanol extracts of the stems and roots showed the strongest inhibition with IC50 values of 204.06 and 216.24 µg/ml, respectively. None of the examined extracts showed any activity against Escherichia coli. while the ethanol extract of the roots and stems showed moderate inhibition for Staphylococcus aureus with minimum inhibitory concentration of 400 µg/ml. Ethanol extract of the roots showed only 30% inhibition of acetylcholine esterase enzyme. The results of anti-oxidant, phenolic content and protective effect against H2O2-induced cytotoxicity assays showed highly correlated data. Ethanol extract of the stems (ORAC value of 1.08 µmol Trolox/mg and phenolic content 44.25 µg GAE/mg) increased the cell viability of H2O2-treated Neuro-2A cells by 28%.

Structure-inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs).

The wide utilization of ginseng provides the high risk of herb-drug interaction (HDI) with many clinical drugs. The inhibition of ginsenosides towards drug-metabolizing enzymes (DMEs) has been regarded as an important reason for herb-drug interaction (HDI). Compared with the deep studies on the ginsenosides' inhibition towards cytochrome P450 (CYP), the inhibition of ginsenosides towards the important phase II enzymes UDP-glucuronosyltransferases (UGTs) remains to be unclear. The present study aims to evaluate the inhibition behavior of ginsenosides towards important UGT isoforms located in the liver and intestine using in vitro methods. The recombinant UGT isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employed as in vitro probe reaction. The results showed that structure-dependent inhibition existed for the inhibition of ginsenosides towards UGT isoforms. To clarify the possibility of in vivo herb-drug interaction induced by this kind of inhibition, the ginsenoside Rg(3) was selected as an example, and the inhibition kinetic type and parameters (K(i)) were determined. Rg(3) competitively inhibited UGT1A7, 2B7 and 2B15-catalyzed 4-MU glucuronidation reaction, and exerted noncompetitive inhibition towards UGT1A8-catalyzed 4-MU glucuronidation. The inhibition parameters (K(i) values) were calculated to be 22.6, 7.9, 1.9, and 2.0µM for UGT1A7, 1A8, 2B7 and 2B15. Using human maximum plasma concentration of Rg(3) (400ng/ml (0.5µM)) after intramuscular injection of 60mg Rg(3), the area under the plasma concentration-time curve (AUC) was extrapolated to increase by 2.2%, 6.3%, 26.3%, and 25% for the co-administered drugs completely undergoing the metabolism catalyzed by UGT1A7, 1A8, 2B7 and 2B15, respectively. All these results indicated that the ginsenosides' inhibition towards UGT isoforms might be an important reason for ginseng-drug interaction.

Isoliquiritigenin showed strong inhibitory effects towards multiple UDP-glucuronosyltransferase (UGT) isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation.

Isoliquiritigenin, a herbal ingredient with chalcone structure, has been speculated to be able to inhibit one of the most drug-metabolizing enzymes (DMEs) UDP-glucuronosyltransferase (UGT). Therefore, the aim of the present study was to investigate the inhibition of isoliquiritigenin towards important UGT isoforms in the liver and intestine, including UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9 and 1A10. The recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as probe reactions. The results showed that 100µM of isoliquiritigenin inhibited the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 by 95.2%, 76.1%, 78.9%, 87.2%, 67.2%, 94.8%, and 91.7%, respectively. The data fitting using Dixon plot and Lineweaver-Burk plot showed that the inhibition of UGT1A1, UGT1A9 and UGT1A10 by isoliquiritigenin was all best fit to the competitive inhibition, and the second plot using the slopes from the Lineweaver-Burk plot versus isoliquiritigenin concentrations was used to calculate the inhibition kinetic parameter (K(i)) to be 0.7µM, 0.3µM, and 18.3µM for UGT1A1, UGT1A9, and UGT1A10, respectively. All these results indicated the risk of clinical application of isoliquiritigenin on the drug-drug interaction and other possible diseases induced by the inhibition of isoliquiritigenin towards these UGT isoforms.

Strong inhibition of celastrol towards UDP-glucuronosyl transferase (UGT) 1A6 and 2B7 indicating potential risk of UGT-based herb-drug interaction.

Celastrol, a quinone methide triterpene isolated from Tripterygium wilfordii Hook F., has various biochemical and pharmacological activities, and is now being developed as a promising anti-tumor agent. Inhibitory activity of compounds towards UDP-glucuronosyltransferase (UGT) is an important cause of clinical drug-drug interactions and herb-drug interactions. The aim of the present study is to investigate the inhibition of celastrol towards two important UDP-glucuronosyltransferase (UGT) isoforms UGT1A6 and UGT2B7. Recombinant UGT isoforms and non-specific substrate 4-methylumbelliferone (4-MU) were used. The results showed that celastrol strongly inhibited the UGT1A6 and 2B7-mediated 4-MU glucuronidation reaction, with 0.9 ± 0.1% and 1.8 ± 0.2% residual 4-MU glucuronidation activity at 100 µM of celastrol, respectively. Furthermore, inhibition kinetic study (Dixon plot and Lineweaver-Burk plot) demonstrated that celastrol noncompetitively inhibited the UGT1A1-mediated 4-MU glucuronidation, and competitively inhibited UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameters (Ki) were calculated to be 0.49 µM and 0.045 µM for UGT1A6 and UGT2B7, respectively. At the therapeutic concentration of celastrol for anti-tumor utilization, the possibility of celastrol-drug interaction and celastrol-containing herbs-drug interaction were strongly indicated. However, given the complicated nature of herbs, these results should be viewed with more caution.

Investigation of UDP-glucuronosyltransferases (UGTs) inhibitory properties of carvacrol.

UDP-glucuronosyltransferases (UGTs), the most important phase II drug metabolizing enzymes (DMEs), could metabolize many drugs and various endogenous substances including bilirubin, steroid hormones, thyroid hormones, bile acids and fat-soluble vitamins. Evaluation of the inhibitory effects of compounds on UGTs is clinically important because inhibition of UGT isoforms could not only result in serious drug-drug interactions (DDIs), but also induce metabolic disorders of endogenous substances. The aim of the present study was to investigate the inhibitory effects of carvacrol on major UGT isoforms. The results showed that carvacrol could inhibit the activity of UGT1A9 with negligible effects on other UGT isoforms. When 4-methylumbelliferone (4-MU) was used as a nonspecific probe substrate and recombinant UGT enzymes were utilized as an enzyme resource, the inhibition of UGT1A9 was best fit to the competitive type and the inhibition kinetic parameter (K(i)) was calculated to be 5.7 µM. Furthermore, another specific probe substrate, propofol, was employed to determine the inhibitory kinetics of UGT1A9, and the results demonstrated that the inhibitory type was noncompetitive. The inhibition kinetic parameter (K(i)) was determined to be 25.0 µM. Because this substrate-dependent inhibition of UGT1A9 might confuse the in vitro-in vivo extrapolation, these in vitro inhibition kinetic parameters should be interpreted with special caution.

Effects of Andrographis paniculata and Orthosiphon stamineus extracts on the glucuronidation of 4-methylumbelliferone in human UGT isoforms.

The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU) by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA) as the cofactor, MgCl(2), cell lysate of respective isoform, and 4MU at the approximate apparent K(m) or S(50) value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 microg/mL and 0.01, 0.10, 1.0, 10 and 50 microg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC(50) 1.70 microg/mL for UGT1A3, 2.57 microg/mL for UGT1A8, 2.82 microg/mL for UGT2B7, 5.00 micorg/mL for UGT1A1, 5.66 microg/mL for UGT1A6, 9.88 microg/mL for UGT1A7 and 15.66 microg/mL for UGT1A10). Both extracts showed less than 70% inhibition of UGT2B15, so the IC(50) values were >50 microg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

Hypolipidemic effect of Pleurotus eryngii extract in fat-loaded mice.

Pleurotus eryngii water extract (PEE), which showed the most significant inhibitory activity against pancreatic lipase in vitro among eight edible mushrooms, was investigated to determine the mechanism of its anti-lipase activity in vitro and its hypolipidemic effect in fat-loaded mice. The inhibitory effects of mushroom extracts on pancreatic lipase activity were examined using 4-methylumbelliferyl oleate (4-MUO) or trioleoylglycerol emulsified with lecithin, gum arabic or Triton X-100 as a substrate. For in vivo experiments, blood samples were taken after oral administration of corn oil and [(3)H]trioleoylglycerol with or without PEE to food-deprived mice. PEE inhibited hydrolysis of 4-MUO and trioleoylglycerol emulsified with lecithin or Triton X-100, but not that of trioleoylglycerol emulsified with gum arabic. PEE suppressed the elevations of plasma and chylomicron triacylglycerol levels after oral administration of corn oil, but had no effect on lipoprotein lipase activity. [(3)H]Trioleoylglycerol absorption was also decreased by administration of PEE. The results of in vitro studies suggest that PEE may prevent interactions between lipid emulsions and pancreatic lipase. The hypolipidemic effect of PEE in fat-loaded mice may be due to low absorption of fat caused by the inhibition of pancreatic lipase.

A synthetic coumarin (4-methyl-7 hydroxy coumarin) has anti-cancer potentials against DMBA-induced skin cancer in mice.

Scopoletin, an alkaloid separated from ethanolic extract of the medicinal plant, Gelsemium sempervirens (Fam: Loganiaceae) has been reported to have anti-cancer potentials. The synthetic coumarin (4-Methyl-7 hydroxy coumarin) derived from resorcinol and ethyl aceto-acetate in presence of concentrated sulphuric acid is structurally close to scopoletin, being a coumarin derivative. Whether this synthetic compound also has anti-cancer potentials has been evaluated in vivo on DMBA (7,12-Dimethylbenz[a]anthracene) induced skin cancer in mice by analyzing results of several cytogenetic endpoints, Comet assay, and fluorescence activated cell sorting (FACS). Further, expressions of signal proteins like Aryl hydrocarbon receptor , p53, PCNA, Akt, Bcl-2, Bcl-xL, Bad, Bax, NF-kappaB Apaf, IL-6, Cytochrome-c, Caspase-3 and Caspase-9 were studied by immunoblot analysis along with histology of skin and immuno-histochemical localization of Aryl hydrocarbon receptor and PCNA in DMBA treated mice vis-a-vis carcinogen treated synthetic coumarin fed mice. Feeding of this synthetic coumarin induced positive modulations in expression of all biomarkers in DMBA administered mice, giving clues on its possible signaling pathway(s) - primarily through down-regulation of Aryl hydrocarbon receptor and PCNA and up-regulation of apoptotic proteins like Bax, Bad, Cytochrome c, Apaf, Caspase-3 and Caspase-9, resulting in an appreciable reduction in growth of papilloma in mice. Therefore, this synthetic coumarin shows promise for use in cancer therapy, particularly in skin cancer.

Silkworm as a model animal to evaluate drug candidate toxicity and metabolism.

To evaluate the feasibility of using the silkworm as a model animal for screening drug candidates, we examined whether the lethal dose of cytotoxic chemicals in silkworm, Bombyx mori, were consistent with those in mammals, and compared the metabolic pathways of these drugs between silkworms and mice. The lethal dose levels of cytotoxic chemicals in silkworms were consistent with those in mammals. We examined the fate of model drugs, 4-methyl umbelliferone, umbelliferone, and 7-ethoxycoumarine, in silkworm larvae. The half-life of 4-methyl umbelliferone in the silkworm larvae hemolymph was 7.0+/-0.1 min, similar to that in mouse blood. In silkworm larvae, 4-methyl umbelliferone was conjugated with glucose, whereas in mammals it is conjugated with glucuronate or sulfate. These results are consistent with a previous report that UDP-glucosyltransferase catalyzes the conjugation of 4-methyl umbelliferone. The glucose-conjugation reaction of 4-methyl umbelliferone was observed in microsomal fractions of fat bodies isolated from silkworms. Furthermore, most umbelliferone and 7-ethoxycoumarine injected into the hemolymph of silkworms was eliminated through the feces in the glucose-conjugated form. These findings suggest that chemicals are metabolized through a pathway common to both mammals and silkworms: reaction with cytochrome P450, conjugation with hydroxylated compounds, and excretion.

Efficacy of 4-methyl-7-hydroxy coumarin derivatives against vectors Aedes aegypti and Culex quinquefasciatus.

4-Methyl-7-hydroxy coumarin is considered as a lead molecule as a biopesticide. Its mono bromo and tribromo derivatives were synthesized. Two more derivatives were synthesized by acylation. Compound 1 (3,6,8-tribromo-7-hydroxy-4-methyl-chromen-2-one) was found to be the most potent against IVth instar larvae of C. quinquefasciatus and A. aegypti the LC50 being 1.49 and 2.23 ppm respectively. It showed 100% larval mortality at 25 ppm against A. aegypti and at 10 ppm against C. quinquefasciatus. Compounds 1 and 2 (3,6,8-tribromo-7-hydroxy-4-methyl-chromen-2'-oxo-2H-chromen-7-yl acetate) showed remarkable ovicidal activity. Significant reduction of 80-85% hatching of eggs of both mosquito species was observed at the highest dose of 100 ppm. The hatched larvae showed 100% mortality in the successive instars. Compounds 3 and 4 (3-bromo-7-hydroxy-4-methyl-chromen-2-one and 3-bromo-4-methyl-2'-oxo-2H-chromen-7-yl acetate) showed moderate activity against both mosquito species.

Novel screening assay for antioxidant protection against peroxyl radical-induced loss of protein function.

Oxidative damage to proteins, implicated amongst other in the etiology and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), results in the loss of specific biological protein function. A simple, sensitive, and cost-effective fluorimetric test to assess the antioxidant capacity of new chemical entities to protect proteins from loss of activity caused by reactive oxygen species (ROS) was developed using alkaline phosphatase (ALP) as model protein. Protein oxidation was induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and the decrease in catalytic activity of ALP to hydrolyze 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) was monitored as a marker of protein degradation. According to their capacity to protect ALP from peroxyl radical-induced activity loss, ten reference antioxidants were divided into three classes, namely efficient (pIC(50) > 5 for quercetin, chlorogenic acid, caffeic acid, mangiferin, and resveratrol), intermediate (4 < pIC(50) < or = 5 for melatonin, trolox, and ascorbic acid), and poor antioxidants (pIC(50) < 4 for glutathione and D-mannitol). Multifunctional drugs, having the ability to interact with several disease-related targets are of interest in PD. Therefore, the capacity of three catechol-O-methyltransferase (COMT) inhibitors, entacapone, nitecapone, and tolcapone to protect ALP from oxidative damage was also investigated and found to be very similar to the most potent reference antioxidants.

Thermodynamic analysis reveals structural rearrangement during the acylation step in human trypsin 4 on 4-methylumbelliferyl 4-guanidinobenzoate substrate analogue.

Human trypsin 4 is an unconventional serine protease that possesses an arginine at position 193 in place of the highly conserved glycine. Although this single amino acid substitution does not affect steady-state activity on small synthetic substrates, it has dramatic effects on zymogen activation, interaction with canonical inhibitors, and substrate specificity toward macromolecular substrates. To study the effect of a non-glycine residue at position 193 on the mechanism of the individual enzymatic reaction steps, we expressed wild type human trypsin 4 and its R193G mutant. 4-Methylumbelliferyl 4-guanidinobenzoate has been chosen as a substrate analogue, where deacylation is rate-limiting, and transient kinetic methods were used to monitor the reactions. This experimental system allows for the separation of the individual reaction steps during substrate hydrolysis and the determination of their rate constants dependably. We suggest a refined model for the reaction mechanism, in which acylation is preceded by the reversible formation of the first tetrahedral intermediate. Furthermore, the thermodynamics of these steps were also investigated. The formation of the first tetrahedral intermediate is highly exothermic and accompanied by a large entropy decrease for the wild type enzyme, whereas the signs of the enthalpy and entropy changes are opposite and smaller for the R193G mutant. This difference in the energetic profiles indicates much more extended structural and/or dynamic rearrangements in the equilibrium step of the first tetrahedral intermediate formation in wild type human trypsin 4 than in the R193G mutant enzyme, which may contribute to the biological function of this protease.

Relative movement and soil fixation of soluble organic and inorganic phosphorus.

There is considerable concern about pollution of surface waters with P. Although most of the research has focused on inorganic P in surface runoff, it has recently become possible to easily follow the fate of soluble organic P forms in soils and waters. Two experiments were performed to compare the relative mobility and soil fixation affinity of orthophosphate monoesters, orthophosphate diesters, and soluble inorganic P. We used three P substrates, 4-methylumbelliferyl phosphate (MUP), deoxyribonucleic acid (DNA), and KH(2)PO(4) in (i) a soil column experiment and (ii) a soil P adsorption test tube experiment. Shortly after columns were prepared, approximately two pore volumes of 0.005 M CaCl(2) were passed through 25 cm length columns containing 10 cm of loamy sand amended with approximately 10 mg P as MUP, DNA, or KH(2)PO(4) above 15 cm of nonamended loamy sand. The total net quantity of 757.8 microg P 2L(-1) of orthophosphate diesters in the leachate from the DNA columns exceeded the net quantity of orthophosphate monoesters in leachate from the MUP columns (4.6 microg P 2L(-1)) and soluble inorganic P from the KH(2)PO(4) columns (34.0 microg P 2L(-1)). Adsorption of soluble organic and inorganic P in the test tube experiment yielded similar results: DNA, containing orthophosphate diesters, had a relatively low affinity for soils. In both experiments, high concentrations of other P compounds were identified in samples treated with organic P substrates, suggesting enzymatic hydrolysis by native soil phosphatase enzymes. These findings indicate that repeated application of organic forms of P could lead to significant leaching of P to ground water.

6,8-Difluoro-4-methylumbiliferyl phosphate: a fluorogenic substrate for protein tyrosine phosphatases.

The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor constants for DIFMUP cleavage were comparable with those of the widely used but less discriminative and practicable substrates, para-nitrophenylphosphate and phosphotyrosine-containing peptides, respectively. Furthermore, the continuous and highly sensitive assay allows fast and accurate investigations of the type, kinetic behavior, and binding mode of small-molecule inhibitors. We discuss the validation of this assay system for various PTPs and its use in inhibitor screening for PTP1B.

DNA damage in healthy term neonate.

BACKGROUND: The process of childbirth is accompanied by an increase in oxidative aggression. AIM: To determine DNA damage and oxidative stress in healthy term neonates at birth. DESIGN: A total of 34 healthy full-term neonates, 22 healthy adults and 20 samples of colostrum from mothers of full-term neonates were examined. The malondialdehyde (MDA), DNA damage, GSH/GSSG ratio and total antioxidant capacity (TAC) in mononuclear cells isolated from umbilical blood and adult peripheral blood were measured. Moreover, the TAC of colostrum was also measured. The protective activity of five natural polyphenols against H(2)O(2)-induced DNA damage in mononuclear cells of umbilical blood was studied. RESULTS: A high level of DNA damage (p<0.001) accompanied with lower TAC (p<0.05) and GSH/GSSG ratio (p<0.001) and with higher level of MDA (p<0.001) in umbilical blood compared with those of healthy adult peripheral blood. The natural polyphenols, 7,8-dihydroxy-4-methyl coumarin, quercetin and resveratrol, are able to protect mononuclear cells of umbilical blood from oxidative attack. However, other two polyphenols, rutin and 7-hydroxy-4-methyl coumarin, do not. The TAC of colostrum is significantly higher than that of umbilical blood (p<0.001). CONCLUSIONS: The DNA oxidative damage in mononuclear cells of umbilical blood as well as other indexes related to redox status provided evidence that a sudden increase in oxygenation exposes the neonate into oxidative stress. Colostrum with a significant high TAC is very important for health care in infants against the oxidative stress.

Coumarins from the aerial part of Halocnemum strobilaceum.

Four known coumarins, coumarin (1), 7-hydroxy-3-methylcoumarin (2), oreoselone (3) and heraclenin (4), were isolated from aerial part of Halocnemum strobilaceum. Their structures were determined by 1 and 2-D NMR techniques.

A rapid fluorescence bioassay for the determination of selenium on agar plates.

The essential trace element selenium (Se) is involved in the form of selenocysteine at the active site of several prokaryotic and eukaryotic proteins called selenoproteins. These proteins have recently attracted attention particularly in relation to their application to human health and new characteristics of the genetic code. We have recently described a selenium bioassay based on a recombinant DNA construct in which the expression of the lac' Z gene in Escherichia coli is proportionally and specifically driven by UGA-directed selenocysteine incorporation. Here we have further developed this bioassay for more rapid and sensitive detection and measurement of selenium that permits screening of the selenium status on agar plates. Again, the inclusion of selenium into the lac'Z-fusion product is reflected by the level of beta-galactosidase activity, which in turn is reflected by the intensity of fluorescence on agar plates. This fluorescing agent is a 4-methylumbelliferyl moiety which is released through the cleavage by the enzyme of 4-methylumbelliferyl-beta-D-galactoside. The intensity of the fluorescence is easily detected by uv irradiation and photographed by polaroid or video cameras.