Liquid chromatography/electrospray ionization mass spectrometry method for the determination of the active metabolite M-1 of suplatast tosilate in human plasma.

A liquid chromatography/electrospray ionization mass spectrometry (LC/ESIMS) method for the determination of 4-(3-ethoxy-2-hydroxypropoxy) acrylanilide (M-1), the active metabolite of suplatast tosilate, in human plasma was established. Plasma samples were extracted with diethyl ether, separated on a C(18) column with a mobile phase of acetonitrile-10 mm ammonium acetate solution containing 0.1% formic acid (28:72, v/v) and detected by ESIMS. The method was linear over the concentration range 0.15-60.0 ng/mL. The lowest limit of quantification was 0.15 ng/mL. The intra- and inter-run relative standard deviations obtained from three validation runs were all less than 8.6%, and the intra- and inter-run relative errors were all less than 3.1%. The method was successfully applied for the evaluation of pharmacokinetic profiles of M-1 in healthy volunteers.

Laboratory simulated dissipation of metsulfuron methyl and chlorimuron ethyl in soils and their residual fate in rice, wheat and soybean at harvest.

Two sulfonylurea herbicides, metsulfuron methyl (Ally 20 WP) and chlorimuron ethyl (Classic 25 WP) were evaluated for their dissipation behaviour in alluvial, coastal saline and laterite soils under laboratory incubated condition at 60% water holding capacity of soils and 30 degrees C temperature was maintained. In field study herbicides were applied twice for the control of grasses, annual and perennials broad leaves weeds and sedges in rice, wheat and soybean to find out the residual fate of both the herbicides on different matrices of respective crops after harvest. Extraction and clean up methodologies for the herbicides were standardized and subsequently analyzed by HPLC. The study revealed that the half-lives of metsulfuron methyl and chlorimuron ethyl ranged from 10.75 to 13.94 d irrespective of soils and doses applied. Field trials with rice, wheat and soybean also revealed that these two herbicides could safely be recommended for application as no residues were detected in the harvest samples.

Chemical control of ambrosia Artemisiifolia on non-crop areas: are there alternatives to glyphosate?

We compared glyphosate, glufosinate and metsulfuron-methyl to control Ambrosia artemisiifolia under non-crop conditions. A laboratory study showed that A. artemisiifolia is an easy-to-wet species and that glufosinate and glyphosate are quickly absorbed by its leaves (nearly 100% in 24 h). Metsulfuron-methyl absorption was slower (about 50% in 24 h) but was strongly promoted by terpenic alcohol and esterified rapeseed oil. In the greenhouse, all three herbicides were efficacious against A. artemisiifolia, with ED50s of <23, 23 and 0.8 g ha(-1) for glufosinate, glyphosate and metsulfuron-methyl, respectively. These results were confirmed on a non-crop area for glufosinate and glyphosate, which at half the registered dose reached high efficacies at both the 4 to 6-node and flowering stages of A. artemisiifolia. By contrast, metsulfuron-methyl showed no efficacy. However, after treatment at the 4- to 6-node stage, new emergence of A. artemisiifolia led to the presence of vigorous plants that bore numerous flowers and produced high levels of pollen. After treatment at the flowering stage, flower production by A. artemisiifolia was not significantly affected, but achene weight was decreased by 60 to 70% and seed viability was only 8 to 13% for the treated plants, as compared to 85% for the control. No significant difference was observed between the two herbicides and between the doses. It is concluded that glufosinate can be an alternative to glyphosate for the chemical control of A. artemisiifolia on non-crop areas. However, with both herbicides, it is difficult to attain the two objectives of reducing seed production and pollen production by means of only one treatment.

Biodegradation of chlorsulfuron and metsulfuron-methyl by Aspergillus niger in laboratory conditions.

Two sulfonylurea herbicides, chlorsulfuron and metsulfuron-methyl, were studied under laboratory conditions, in order to elucidate the biodegradation pathway operated by Aspergillus niger, a common soil fungus, which is often involved in the degradation of xenobiotics. HPLC-UV was used to study the kinetic of degradation, whereas LC-MS was used to identify the metabolites structure. In order to avoid the chemical degradation induced by a decrease in pH, due to the production of citric acid by the fungus, the experiments were performed in a buffered neutral medium. No significant degradation for both compounds was observed in mineral medium with 0.2% sodium acetate. On the contrary, in a rich medium, after 28 days the degradations, chemical degradation excluded, were about 30% for chlorsulfuron and 33% for metsulfuron-methyl. The main microbial metabolites were obtained via cleavage of the sulfonylurea bridge. In addition the fungus seems to be able to hydroxylate the aromatic ring of chlorsulfuron. In the case of metsulfuron-methyl the only detected metabolite was the triazine derivative, while the aromatic portion was completely degraded. Finally, the demethylation of the methoxy group on the triazine ring, previously observed with a Pseudomonas fluorescens strain, was not observed with A. niger.

Vaginal-cervical epithelial permeability decreases after menopause.

OBJECTIVE: To determine the effects of menopause (aging and E) on vaginal-cervical epithelial paracellular permeability. DESIGN: Experimental, basic clinical research. SETTING: Academic research environment. PATIENT(S): Premenopausal, perimenopausal, and postmenopausal women, aged 35-65 years. INTERVENTION(S): Primary to tertiary cultures of normal human ectocervical epithelial cells on filters. Cells were outgrown from surgically discarded ectocervical minces. MAIN OUTCOME MEASURE(S): Changes in paracellular permeability were determined as changes in transepithelial electrical conductance and pyranine permeability. RESULT(S): [1] Levels of transepithelial electrical conductance and pyranine permeability decreased as women's age advanced. [2] Removal of E from the culture medium decreased paracellular permeability. Treatment of cells in vitro with 10 nmol/L 17beta-E2 increased transepithelial electrical conductance and pyranine permeability, but the effects were additive to the age-related decrease in permeability. [3] Coadministration of 100 nmol/L tamoxifen blocked the E increase in paracellular permeability in cells of both premenopausal and postmenopausal women. CONCLUSION(S): [1] Aging and E deficiency decrease independently vaginal-cervical epithelial paracellular permeability. [2] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is mediated by the E receptor. [3] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is masked by age-related increase in the tight junctional resistance, leading to overall decrease in paracellular permeability.

Uptake of a natural surfactant and increased delivery of small organic anions into type II pneumocytes.

The uptake of natural lung surfactant into differentiated type II cells may be used for the targeted delivery of other molecules. The fluorescent anion pyranine [hydroxypyren-1,3,6-trisulfonic acid, sodium salt (HPTS)] was incorporated into a bovine surfactant labeled with [3H]dipalmitoylphosphatidylcholine ([3H]DPPC). The uptake of [3H]DPPC and of HPTS increased with time of incubation and concentration, decreased with the size of the vesicles used, and was stimulated by 8-bromo-cAMP and partially inhibited by hypertonic sucrose. However, the amount of HPTS uptake was approximately 100 times smaller than that of [3H]DPPC. This large difference was due to a more rapid regurgitation of some of the HPTS from the cells but not to leakage from the surfactant before uptake. The acidification of the internalized surfactant increased linearly over 90 min to 7.13, and after 24 h, a pH of 6.83 was measured. In conclusion, after internalization of a double-labeled natural surfactant, the lipid moieties were accumulated in relation to the anions, which were targeted to a compartment not very acidic and in part rapidly expelled from the cells.

Intracellular pH-dependent efflux of the fluorescent probe pyranine in the yeast Yarrowia lipolytica.

8-Hydroxypyrene-1,3,6-trisulfonic acid (pyranine) can be used as a vital intracellular pH (pH(i)) indicator. In the yeast Yarrowia lipolytica, a partial efflux of the probe was detected by using the pH-independent wavelength of 415 nm. A simplified correction of the fluorescent signals was applied, enabling to show for this species a good near-neutral pH(i) maintenance capacity in a pH 3.9 medium. Octanoic acid, which is known to have toxic effects on yeast, decreased the pH(i) and increased the 260-nm-absorbing compounds leakage. However, this acid inhibited the fluorescent probe efflux linearly with its concentration suggesting a pH(i)-dependent efflux of pyranine from cells.

Time-resolved study of the inner space of lactose permease.

Pyranine (8-hydroxy pyrene-1,3,6-trisulfonate) is a commonly used photoacid that discharges a proton when excited to its first electronic singlet state. Follow-up of its dissociation kinetics reveals the physicochemical properties of its most immediate environment. At vanishing ionic strength the dye adsorbs to the Escherichia coli lactose permease with stoichiometry of 1:1 and an association constant of 2.5 x 10(5) M(-1). The reversal of the binding at high ionic strength and the lower pK value of the bound dye imply that positive charge(s) stabilize the dye in its site. The fluorescence decay curve of the bound dye was measured by time-correlated single photon counting and the measured transient was subjected to kinetic analysis based on the geminate recombination model. The analysis indicated that the binding domain is a cleft (between 9 and 17 A deep) characterized by low activity of water (a((water)) = 0.71), reduced diffusivity of protons, and enhanced electrostatic potential. The binding of pyranine and a substrate are not mutually exclusive; however, when the substrate is added, the dye-binding environment is better solvated. These properties, if attributed to the substrate-conducting pathway, may explain some of the forces operating on the substrate in the cavity. The reduced activities of the water strips the substrate from some of its solvation water molecules and replace them by direct interaction with the protein. In parallel, the lower dielectric constant enhances the binding of the proton to the protein, thus keeping a tight seal that prevents protons from diffusing.

5,6-Dihydropyran-2-ones possessing various sulfonyl functionalities: potent nonpeptidic inhibitors of HIV protease.

On the basis of previous SAR findings and molecular modeling studies, a series of compounds were synthesized which possessed various sulfonyl moieties substituted at the 4-position of the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents were added in an attempt to fill the additional S(3)' pocket and thereby produce increasingly potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of selected compounds were synthesized. All analogues in the study displayed decent binding affinity to HIV protease, and several compounds were shown to possess very good antiviral efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide and sulfonate moieties were filling the S(3)' pocket of the enzyme. However, the additional substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the currently marketed protease inhibitors.

Estrogen modulates paracellular permeability of human endothelial cells by eNOS- and iNOS-related mechanisms.

Estradiol had a biphasic effect on permeability across cultures of human umbilical vein endothelial cells (HUVEC): at nanomolar concentrations it decreased the HUVEC culture permeability, but at micromolar concentrations it increased the permeability. The objective of the present study was to test the hypothesis that the changes in permeability were mediated by nitric oxide (NO)-related mechanisms. The results revealed dual modulation of endothelial paracellular permeability by estrogen. 1) An endothelial NO synthase (eNOS)-, NO-, and cGMP-related, Ca2+-dependent decrease in permeability was activated by nanomolar concentrations of estradiol, resulting in enhanced Cl- influx, increased cell size, and increases in the resistance of the lateral intercellular space (RLIS) and in the resistance of the tight junctions (RTJ); these effects appeared to be limited by the ability of cells to generate cGMP in response to NO. 2) An inducible NO synthase (iNOS)- and NO-related, Ca2+-independent increase in permeability was activated by micromolar concentrations of estradiol, resulting in enhanced Cl- efflux, decreased cell size, and decreased RLIS and RTJ. We conclude that the net effect on transendothelial permeability across HUVEC depends on the relative contributions of each of these two systems to the total paracellular resistance.

Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging.

Although used extensively for the measurement of intracellular pH, derivatives of fluorescein such as 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have suboptimal sensitivity and can generate toxic photoproducts. These limitations can be overcome using the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine), which has improved spectroscopic properties. However, the use of pyranine has been limited by the difficulties encountered in delivering this highly hydrophilic dye to the cell interior. We describe a strategy for intracellular delivery of pyranine based on the reversible activation of purinergic P2x7 receptors, which allow permeation of the dye into otherwise intact cells. When loaded into J774 or RAW cells by this method, pyranine is not only more sensitive than BCECF (the dynamic range is approximately 7-fold greater), but is retained better and is less toxic. Pyranine was distributed throughout the cytosol but was not detectable in endomembrane compartments. Repeated illumination resulted in blebbing and loss of functional responsiveness of cells loaded with BCECF, whereas comparably irradiated cells loaded with pyranine remained healthy and responsive. Pyranine can also be loaded into cells not expressing P2x7 receptors by brief exposure to a hypotonic solution. The properties of cells labeled by this method are similar to those loaded via purinergic receptors and compare favorably with those of BCECF-loaded cells. Pyranine thus provides a useful alternative to fluorescein derivatives for the measurement of intracellular pH, particularly when using the high excitation intensities required for microscopic digital imaging.

Osmolar changes regulate the paracellular permeability of cultured human cervical epithelium.

Extracellular nucleotides induce a biphasic change in the transepithelial electrical conductance (GT) of human cervical cells grown on filters: a rapid increase (phase I) followed by a sustained decrease (phase II). To probe the involvement of the intercellular space, its magnitude was varied by manipulating cell volume through changes in extracellular osmolarity. Under baseline conditions [GT = 115 mS/cm2 (approximately 9 omega.cm2)] and during phase II, hypertonic challenges resulted in an increase in GT (0.98% .mosmol-1.l-1 and 0.73%.mosmol-1.l-1, respectively). However, a hypertonic challenge during phase I decreased GT (-0.16%.mosmol-1.l-1). Hypotonic challenges decreased GT during baseline, phase I, and phase II conditions by -1%.mosmol-1.l-1. Similar trends were observed with regard to pyranine permeability. Reduction of extracellular calcium increased GT, abrogated the phase II effect of extracellular ATP, and reversed the effect of a hypertonic challenge. The additive nature of the permeability changes in response to osmotic challenges and to ATP during phase II suggests that different sites are involved in each response, i.e., the resistance of the intercellular space changes with osmolarity and that of the tight junction during phase II.

Suppressive effects of IPD-1151T (suplatast-tosilate) on induction of mast cells from normal mouse splenocytes.

The influence of IPD-1151T (suplatast-tosilate) on murine mast cell induction was examined by in vitro cell culture. Splenocytes from BALB/c mice suspended in RPMI-1640 medium supplemented with interleukin-3 were cultured in the presence or absence of IPD-1151T. Half of the total volume of the medium was changed every 4-5 days. The number of mast cells increased with culture time and reached a peak on the 16th day when the cells were cultured without IPD-1151T. However, the growth of mast cells from the splenocyte culture was dose dependently inhibited by IPD-1151T. Furthermore, IPD-1151T inhibited the proliferation of mast cell progenitor cells, IC-2, but not that of splenocytes or mature mast cells.

ATP decreases acutely and reversibly transport through the paracellular pathway in human cervical cells.

We studied the effect of ATP on transepithelial transport through the paracellular pathway in human cervical cells. Transepithelial conductance and transepithelial permeability (determined from the measurements of unidirectional flux of inert molecules) were measured in Caski cells grown on permeable support. Transepithelial conductance was 55.9 +/- 17.7 mS/cm2 and permeability was 12.5 +/- 2.7 x 10(-6) cm/s for a 0.51-kDa probe. Addition of ATP to the medium decreased acutely and reversibly the conductance and the permeability to probes between 0.18 and 10 kDa by 23-31% in a dose-related fashion; the 50% effective concentration was 1 microM, with a maximal effect at 5-10 microM extracellular ATP. The ATP effect was observed regardless of the pressure gradient across the epithelium. These results indicate that extracellular ATP in micromolar concentrations decreases acutely and reversibly the permeability through the paracellular pathway in cervical cells, possibly by affecting the permeability of the tight junctions and the resistance of the intercellular space. On the basis of these data, we speculate that ATP may play a role in the regulation of solutes and fluid transport across the cervical epithelium in vivo.