Interactions of an acidic cyclooctapeptide with metal ions: microcalorimetric and fluorescence analyses

Abstract: The metal‐ion binding preferences of an acidic amphipathic cyclopeptide, cyclo[d ‐Leu‐Leu‐d ‐Leu‐Trp‐(d ‐Glu‐Glu)₂] (CP), was studied by isothermal titration calorimetry and steady‐state fluorescence spectroscopy. CP adopted a partial beta structure, and variable temperature circular dichroism showed small secondary structural changes over the temperature range from 5 °C to 95 °C. The peptide did not bind alkali or alkaline earth metal ions but exhibited selectivity for some divalent transition metal ions (with association constants K_Cu2+ 4.5 × 10⁴ m ^?1, K_Zn2+ 1.6 × 10⁵ m ^?1, K_Cd2+ 1.3 × 10⁴ m ^?1, K_{Hg2+ (1)} 2.2 × 10⁶ m ^?1, and K_{Hg2+ (2)} 6.5 × 10³ m ^?1), for Pb²⁺ (2.0 × 10⁵ m ^?1), and a trivalent Group III metal, Al³⁺ (1.6 × 10⁵ m ^?1). The thermodynamic data show that the interaction between CP and these metal ions are spontaneous and entropically driven. A large range of binding enthalpies coupled with a smaller range of binding free energies of CP for these metal ions indicate an entropy–enthalpy compensation dependent on the ionic size of participating metal ions. The interaction of Pb²⁺, Hg²⁺, and Cu²⁺ with CP in aqueous solution specifically modulates the fluorescence emission properties of CP. The results of this study show that CP exhibits selectivity in metal‐ion binding, which is reflected in its fluorescence spectra. The observed trends can be useful for the design of heavy‐metal sensors based on fluorophore‐tagged acidic cyclopeptides.     

Effects of pH and phosphate on the oxidation of iron in aqueous solution

Studies have been initiated to evaluate the catalytic effect of monohydrogen phosphate ions on the oxidation of ferrous (Fe²⁺) to ferric (Fe³⁺) ions in an aqueous solution under atmospheric oxygen conditions. The reactions were performed with an initial concentration of 1 × 10^?4 M ferrous sulfate in solutions containing varying concentrations of phosphate buffer (0.005–0.0175 M) over the pH range of 6.6–7.1. The final ionic strength of the solutions were adjusted to 0.1 M with sodium chloride and the temperature was kept constant at 25 ± 0.5 °C. The rates of oxidation reactions were measured by following the increase in UV absorbance due to the formation of ferric ion in solution. The reactions appeared to follow pseudo-first-order kinetics and were very prone to catalysis by monohydrogen phosphate at any given pH. H₂PO₄^? seemed to have no effect on the reaction. HPO_s^2? was the sole catalytic species with a second-order rate constant of 116.74 M^?1 · min^?1. The buffer independent pH-rate profile showed a sigmoidal behavior with the pseudo-first-order rate constant increasing with increasing pH. The sigmoidal nature of the experimental pH-rate profile could possibly suggest a change in the reactivity of the oxidizing species which might follow complex kinetics. The effects of ionic strength and temperature on the reaction rates were also evaluated.     

A novel flow‐injection chemiluminescence method for determination of andrographolide in andrographis tablets

A novel method for determination of andrographolide using flow‐injection chemiluminescence (FI‐CL) analysis is described in this paper. The chemiluminescence intensity of the solution was enhanced proportionally while the concentration of andrographolide increased. Under the selected experimental conditions, the calibration curve of andrographolide was linear within the range of 0.2 to 35.0 µg mL^?1 with a linear equation of ΔI = 23.391x (µg mL^?1) + 34.191, R² = 0.9965. The detection limit (3σ) was 7.42 × 10^?2 µg mL^?1. At the case of continuous determination of andrographolide, the relative standard deviation (RSD, n = 11) was less than 1.82%. The method has been successfully applied to the determination of andrographis tablets with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

Influence of iron on modulation of the antioxidant system in rat brains exposed to lead

The objective of this study was to evaluate markers of oxidative stress in the brains of rats exposed to lead acetate (Pb(C₂H₃O₂)₂), either associated or not associated with ferrous sulfate (FeSO₄). A total of 36 weaning rats (Rattus norvegicus) were divided into 6 groups of six animals and exposed to lead acetate for six weeks. In the control group (control), the animals received deionized water. The Pb260 and Pb260 + Fe received 260 µM lead acetate, and the Pb1050 and Pb1050 + Fe received 1050 µM lead acetate. The Pb260 + Fe and Pb1050 + Fe were supplemented with 20 mg of ferrous sulfate/Kg body weight every 2 days. Group Fe received deionized water and ferrous sulfate. The rat brains were collected to analyze the enzymatic activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and the concentration of reduced glutathione (GSH), lipid peroxidation (TBARS), and total antioxidant substance (TAS) (DPPH^? technique). The activity of SOD and GPx in the experimental groups decreased compared to the control, together with the concentration of GSH (p < 0.05). For CAT analysis, SOD tended to increase in concentration in the experimental groups without a concomitant exposure to FeSO₄, whereas GPx showed a slight tendency to increase in activity compared to the control. For TAS‐DPPH^?, there was a decrease in the experimental groups (p < 0.05). According to the results, SOD, GPx, and GSH were affected by lead acetate and exposure to ferrous sulfate changed this dynamic. However, further studies are needed to verify whether ferrous sulfate acts as a protectant against the toxic effects of lead. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 813–822, 2017.     

Chromatographic determination of drugs of abuse in vitreous humor using solid‐phase extraction

A simple method is presented for the simultaneous determination of morphine, 6‐acetylmorphine, codeine, cocaine, benzoylecgonine, cocaethylene, methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in vitreous humor by high‐performance liquid chromatography with photodiode array detector after solid‐phase extraction with Oasis® HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerra® RP8 column (250 × 4.6 mm i.d., 5 µm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradient mode. A linear response from the detector was obtained within the concentration range of 0.1–4 µg ml^?1, with correlation coefficients higher than 0.99. The limits of detection were lower than 30 ng ml^?1 for all the drugs studied, the coefficients of variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs except for EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming from individuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in five cases and heroin in three cases. Average concentrations of 0.30 µg ml^?1 for morphine, 0.24 µg ml^?1 for 6‐acetylmorphine, 0.10 µg ml^?1 for codeine, 0.81 µg ml^?1 for cocaine, 1.26 µg ml^?1 for benzoylecgonine, 0.15 µg ml^?1 for cocaethylene, 0.11 µg ml^?1 for methadone and 0.68 µg ml^?1 for EDDP were obtained. Copyright © 2012 John Wiley & Sons, Ltd.     

Spectrophotometric determination of penicillins in pure and pharmaceutical formulations using Folin‐Ciocalteu reagent

A new combination of time, temperature, and alkali is described for the spectrophotometric determination of amoxicillin and ampicillin using Folin‐Ciocalteu reagent. The method is based on the development of blue‐coloured product due to the reduction of tungstate and/or molybdate in Folin‐Ciocalteu reagent by amoxicillin and ampicillin in alkaline medium. The chromogenic reaction has λ_max at 720 and 740 nm with molar absorptivity 1.6295 × 10⁴ and 0.1085 × 10⁴ l mol^?1 cm^?1 in the Beer's Law range 2–10 µg mL^?1 and 10–70 µg mL^?1 for amoxicillin and ampicillin, respectively. The method is reproducible, quick, inexpensive, and particularly helpful in determining the drug content in commercial dosage forms. Copyright © 2010 John Wiley & Sons, Ltd.     

Cardiovascular effects induced by N-(4'-dihydro)-piperoylthiomorpholine in normotensive rats

Objectives We have tested the cardiovascular effects of N‐(4′‐dihydro)‐piperoylthiomorpholine (LASSBio 365) on rats using an in‐vivo and in‐vitro approach. Methods LASSBio 365 (0.025, 0.05, 0.1, 0.25, 0.5 or 1 mg/kg, randomly injected) was administered to conscious unrestrained rats and the mean arterial pressure and heart rate were measured. The effects of LASSBio 365 (3 × 10^?6–3 × 10^?4m ) on rat isolated aortic rings with and without endothelium were investigated. Key findings LASSBio 365 induced a dose‐dependent decrease in mean arterial pressure and heart rate (ED50 = 158 ± 53 µg/kg). The effects evoked by LASSBio 365 (0.5 mg/kg) were inhibited by pretreatment with atropine. In anaesthetized rats, electrocardiogram recordings revealed second/third degree sinoatrial and atrioventricular blockade induced by the compound, which were completely inhibited after cardiac muscarinic blockade or cervical bilateral vagotomy. In rat isolated aortic rings, LASSBio 365 (3 × 10^?6–3 × 10^?4m ) was capable of antagonizing the contractile effects induced by phenylephrine (1 µm ) or KCl (80 mm ) (IC50 = 107 ± 6; 92 ± 6 µm , respectively). This effect was not inhibited after removal of the vascular endothelium (IC50 = 84 ± 4; 92 ± 10 µm , respectively). LASSBio 365 (10^?6–10^?4m ) antagonized CaCl₂‐induced contractions in a concentration‐dependent manner. Furthermore, LASSBio 365 (98 µm ) inhibited contractions produced by noradrenaline (1 µm ), but not those induced by caffeine (20 mm ). Conclusions These results suggested that LASSBio 365 produced negative chronotropism and reduced peripheral resistance that were probably due to the stimulation of cardiac muscarinic pathways. Peripheral vasodilation was probably linked to voltage‐dependent Ca²⁺‐channel blockade and/or specific inhibition of Ca²⁺ release from noradrenaline‐sensitive intracellular stores.     

Intestinal transport of bis(12)-hupyridone in Caco-2 cells and its improved permeability by the surfactant Brij-35

The objective of the present study was to elucidate the mechanisms of intestinal transport of bis(12)‐hupyridone (B12H) to predict its oral bioavailability. The effect of the B12H concentration and the contribution of the drug efflux transporters, P‐glycoprotein (P‐gp or ABCB1) and multidrug resistance‐associated proteins (MRPs or ABCC) on B12H absorption were measured and evaluated using the human intestinal epithelial Caco‐2 cell monolayer in the presence of transporter inhibitors. The results indicated that B12H was absorbed in a dose‐dependent manner at concentrations ranging from 132 to 264 µM . However, only apical efflux was observed in the directional transport studies for B12H below 88 µM (P_app(AP‐to‐BL): virtually zero; P_app(BL‐to‐AP): 1.591 ± 0.071 × 10^?5 cm s^?1). P‐gp and mixed P‐gp/MRP inhibitors significantly increased the absorptive transport (P_app(AP‐to‐BL)) to 0.619 ± 0.018 × 10^?5 and 0.608 ± 0.025 × 10^?5 cm s^?1, respectively, while decreasing secretory transport (P_app(BL‐to‐AP)) by >75%. A multiple‐MRP inhibitor, probenecid, increased the P_app(AP‐to‐BL) to 0.329 ± 0.015 × 10^?5 cm s^?1 while decreasing the P_app(BL‐to‐AP) by 50%. Another multiple‐MRP inhibitor, indomethacin, only modestly decreased the P_app(BL‐to‐AP) by ～30% and had no effect on the absorptive transport (P_app(AP‐to‐BL): virtually zero). In addition, the effect of various pharmaceutical excipients (e.g. Pluronic F‐68, Tween‐80 and Brij‐35) on B12H transport was determined and compared. Among them, Brij‐35 effectively enhanced B12H absorption at a concentration lower than its critical micelle concentration (CMC, 60 µM ). Therefore, Brij‐35 can be used as a potential enhancer to improve intestinal absorption of B12H for oral administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Inhibitory effect of 1,8-cineole on guinea-pig airway challenged with ovalbumin involves a preferential action on electromechanical coupling

• 1 1,8‐Cineole is a terpenoid constituent of essential oils with anti‐inflammatory properties. It reduces the neural excitability, functions as an antinociceptive agent and has myorelaxant actions in guinea‐pig airways. The aim of the present study was to investigate the mechanism underlying the myorelaxant effects of 1,8‐cineole in guinea‐pig isolated trachea from either naïve guinea‐pigs or ovalbumin (OVA)‐sensitized animals subjected to antigenic challenge.
• 2 Isometric recordings were made of the tone of isolated tracheal rings. Rings with an intact epithelium relaxed beyond basal tone in the presence of 1,8‐cineole (6.5 × 10^?6 to 2 × 10^?2 mol/L) in a concentration‐dependent manner (P < 0.001, anova ) with a pD₂ value of 2.23 (95% confidence interval 2.10–2.37). Removal of the epithelium or pretreatment of intact tissue for 15 min with 50 µmol/L N^G‐nitro‐l ‐arginine methyl ester, 5 mmol/L tetraethylammonium, 0.5 µmol/L tetrodotoxin or 5 µmol/L propranolol did not alter the potency (pD₂) or the maximal myorelaxant effect (E_max) of 1,8‐cineole.
• 3 1,8‐Cineole also significantly decreased the Schultz‐Dale contraction induced by OVA, mainly in preparations from OVA‐sensitized animals submitted to antigen challenge. 1,8‐Cineole decreased tracheal hyperresponsiveness to KCl and carbachol caused by antigen challenge and almost abolished the concentration–response curves to KCl, whereas it had little effect on the concentration–response curves to carbachol. Under Ca²⁺‐free conditions and in the presence of 10^?4 mol/L acetylcholine, neither 1,8‐cineole (6.5 × 10^?3 mol/L) nor verapamil (1 × 10^?5 mol/L) affected Ca²⁺‐induced contractions, but they almost abolished Ba²⁺‐induced contractions.
• 4 In conclusion, the findings of the present study show that 1,8‐cineole is a tracheal myorelaxant that acts preferentially on contractile responses elicited electromechanically.

     

Electrochemical study of spiramycin and its determination in pharmaceutical preparation

Spiramycin (SPY) is a medium‐spectrum antibiotic with high effectiveness against Gram‐positive bacteria. The voltammetric behaviour of spiramycin was studied using differential pulse polarography (DPP) and square wave polarography (SWP). The drug in Britton‐Robinson buffer (pH 11.5) is reduced at ? 1.45 V, giving rise to a well‐defined cathodic peak using hanging mercury drop electrode (HMDE) versus Ag/AgCl electrode. This peak is attributed to the reduction of the aldehyde group. The results proved that the reduction of SPY is an irreversible diffusion‐controlled process. The diffusion current‐concentration relationship was shown to be rectilinear over the range of 20–80 and 0.8–80 µg ml^?1 using DPP and SWP modes, respectively, with detection limit of 8.5 µg ml^?1 (1.01 × 10^?5 M) and 0.46 µg ml^?1 (5.46 × 10^?7 M) for DPP and SWP modes, respectively. A mechanism is postulated for the reduction of SPY. The proposed techniques were successfully applied to the determination of the studied compound either in pure form or in its formulation. Copyright © 2010 John Wiley & Sons, Ltd.     

Indirect fibre‐optic colorimetric determination of ascorbic acid using 2‐(5‐bromo‐2‐pyridylazo)‐5‐diethylaminophenol and cloud point extraction

A new method has been developed for the indirect determination of ascorbic acid (AA) in commercial syrup preparations based on cloud point extraction (CPE) separation and preconcentration, and determination by molecular absorption spectrometry. The colorimetric method was based on the reduction of Fe(III) to Fe(II) and complexation of Fe(II) with 2‐(5‐bromo‐2‐pyridylazo)‐5‐diethylaminophenol (Br‐PADAP), followed by its extraction into Triton X‐114. Selectivity of the method was increased with the use of EDTA as a masking agent. The absorbance was measured at 742 nm. Various influencing factors on the separation and preconcentration of AA have been investigated systematically, and the optimized operation conditions were established. The proposed method allows the determination of AA in the range 5–200 µg L^?1 with a relative standard deviation of 3.0%. The detection limit was found to be 0.9 µg L^?1 for AA. This method has been applied to the determination of ascorbic acid in commercial pharmaceutical preparations. Copyright © 2011 John Wiley & Sons, Ltd.     

Phosphoenolpyruvate carboxykinase: Effects of the hyperglycaemic agent 3-aminopicolinic acid

Chelation by 3-aminopicolinic acid of Fe²⁺, Co²⁺ and Mn²⁺ has been measured spectrophotometrically. With Fe²⁺ or Co²⁺, 3-aminopicolinic acid inhibited phosphoenolpyruvate carboxykinase at low metal-ion concentrations. Activation was not observed. The enzyme appears not to bind 3-aminopicolinic acid. 3-Aminopicolinic acid protects phosphoenolpyruvate carboxykinase from inactivation by ferrous ions. Two models are suggested, which could account for activation of gluconeogenesis due to chelation of metal-ions by 3-aminopicolinic acid: (a) phosphoenolpyruvate carboxykinase may be in dynamic equilibrium between inactivation by ferrous ions and reactivation by thiol compounds; and (b) metal-ions may promote product-inhibition by phosphoenolpyruvate, which chelation could alleviate.     

Long‐term exposures to di‐n‐butyl phthalate inhibit body growth and impair gonad development in juvenile Murray rainbowfish (Melanotaenia fluviatilis)

The aim of the present study was to evaluate whether long‐term exposures to environmentally relevant concentrations of di‐n‐butyl phthalate (DnBP) disrupt the reproduction‐based endpoints in juvenile Murray rainbowfish (Melanotaenia fluviatilis). Fish were exposed to 5, 15 or 50 µg l^?1 DnBP for 30, 60 and 90 days each, and the effects on survival, body growth, whole‐body concentrations of sex steroid hormones and gonadal development were investigated. The lowest observed effective concentration to affect the condition factor after 90 days was 5 µg l^?1. Complete feminization of the gonad was noted in fish exposed to 5 µg l^?1 for 90 days and to 15 and 50 µg l^?1 of DnBP for 30 or 60 days. After 90 days of exposure to DnBP, the ovaries were regressed and immature as opposed to the control fish which were in early‐vitellogenic stage. Testes, present only in fish exposed to 5 µg l^?1 of DnBP for 30 or 60 days, were immature in comparison to the control fish that contained testes in the mid‐spermatogenic phase. The E2/11‐KT ratio was significantly higher only after exposures to 5 µg l^?1 DnBP for 90 days and 50 µg l^?1 DnBP for 30 days. Our data suggest that exposures to 5 µg l^?1 DnBP for 30 days did not have profound effects on body growth and gonadal differentiation of fish. However, 30 days of exposure to 15 µg l^?1 could interfere with the gonad development and to 50 µg l^?1 could compromise the hormonal profile of juvenile fish. Copyright © 2014 John Wiley & Sons, Ltd.     

Diphenyl diselenide in its selenol form has dehydroascorbate reductase and glutathione S‐transferase‐like activity dependent on the glutathione content

Objectives The antioxidant action of diphenyl diselenide ((PhSe)₂) is attributed to the mechanism by which (PhSe)₂ has pharmacological activity. Although (PhSe)₂ has glutathione peroxidase mimetic activity, the exact mechanism involved in its antioxidant effect has not yet been completely elucidated. In the present study, mechanisms involved in the antioxidant property of (PhSe)₂ (1–50 µm ) were investigated. Methods Dehydroascorbate (DHA) reductase‐ and glutathione S‐transferase (GST)‐like activity, 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activity and the protection against the oxidation of Fe²⁺ were evaluated. Key findings (PhSe)₂ at concentrations equal to, or greater than, 5 µm showed DHA reductase‐ and GST‐like activity. (PhSe)₂ was not a scavenger of DPPH or ABTS radicals and did not protect against the oxidation of Fe²⁺. Conclusions These results clearly indicated that DHA reductase‐ and GST‐like activity are the mechanisms involved in the antioxidant effect of (PhSe)₂.     

Determination of pethidine hydrochloride using potentiometric coated graphite and carbon paste electrodes

A new approach for lowering the detection limit of a pethidine ion‐selective electrode is presented. A coated graphite (CGE) and carbon paste (CPE) electrodes for pethidine ions based on pethidine‐phosphotungstate (PD‐PT) as ion‐pair complex are described. The sensors exhibit a Nernstian slope of 58.1 and 54.2 mVdecade^?1 for pethidine ion over a wide concentration range from 2.6 × 10^?7 to 1.0 × 10^?2 M and 2.1 × 10^?6 to 1.0 × 10^?2 M with a detection limit of 1.8 × 10^?7 M and 7.3 × 10^?7 M for pethidine coated graphite (PD‐CGE) and pethidine carbon paste electrode (PD‐CPE), respectively. These sensors exhibited a fast response time (about 5–8 s) and good stability. The standard electrode potentials, E^o, were determined at different temperatures and used to calculate the isothermal temperature coefficient (dE^o/dT) of the PD‐CGE and PD‐CPE, which was 0.0062 and 0.0071 V/ °C, respectively. Selectivity coefficients, determined by matched potential method (MPM) and separate solution method (SSM), showed high selectivity for pethidine hydrochloride (PDCl) over a large number of inorganic cations, organic cations, sugars, urine components, and some common drug excipients. The sensors were applied for determination of PDCl in ampoule and in spiked urine samples using potentiometric determination, standard addition and the calibration curve methods. The results obtained were satisfactory with excellent percentage recovery comparable and sometimes better than those obtained by other routine methods for the assay. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro metabolism of the epoxide substructure of cryptophycins by cytosolic glutathione S-transferase: Species differences and stereoselectivity

The enzyme kinetics of the glutathione (GSH) conjugation of cryptophycin 52 (C52, R-stereoisomer) and cryptophycin 53 (C53,?S-stereoisomer) by cytosolic glutathione S-transferases (cGSTs) from human, rat, mouse, dog and monkey liver were studied. V_max, K_m, and CL_int values for glutathione conjugation of C52 (R-stereoisomer) were 0.10?±?0.01?nmol?min^?1?mg^?1, 3.24?±?0.23?µM, and (3.15?±?0.09)?×?10^?2?ml?min^?1?mg^?1, respectively, in human cytosol. Due to limited solubility relative to the K_m, only CL_int values were determined in rat ((7.76?±?0.10)?×?10^?2?ml?min^?1?mg^?1) and mouse ((7.61?±?0.50)?×?10^?2?ml?min^?1?mg^?1) cytosol. Enzyme kinetic parameters could not be determined for C53 (S-stereoisomer). Microsomal GSH conjugation in human, rat, and mouse was attributed to cytosolic contamination. No GSH conjugation was seen in any biological matrix from dog or monkey. There was little GSH conjugation of C53 by cytosol or microsomes from any species. The metabolism of C52 and C53 by epoxide hydrolase was also investigated. No diol product was observed in any biological matrix from any species. Thus, cGSTs are primarily responsible for C52 metabolism.     

Effects of Antrodia camphorata on viability,apoptosis, [Ca2+]i,and MAPKs phosphorylation in MG63 human osteosarcoma cells

The present study explored the effect of Antrodia camphorata (AC) on viability, apoptosis, mitogen‐activated protein kinases (MAPKs) phosphorylation, and Ca²⁺ regulation in MG63 human osteosarcoma cells. AC (25–50 µg/ml) did not affect cell viability, but at 100–200 µg/ml decreased viability and induced apoptosis in a concentration‐dependent manner. AC at concentrations of 25–200 µg/ml did not alter basal [Ca²⁺]_i, but at 25 µg/ml decreased [Ca²⁺]_i increases induced by ATP, bradykinin, histamine, and thapsigargin. ATP, bradykinin, and histamine increased cell viability while thapsigargin decreased it. AC (25 µg/ml) pretreatment failed to alter bradykinin‐ and thapsigargin‐induced effects on viability, but potentiated ATP‐ and histamine‐induced increases in viability. Immunoblotting showed that MG63 cells did not have background phospho‐JNK and phospho‐p38 mitogen‐activated protein kinases (MAPKs); and AC did not induce the phosphorylation of these two MAPKs. Conversely, the cells had significant background phospho‐ERK MAPK that was inhibited by 200 µg/ml AC. The ERK‐specific inhibitor PD98059 also induced cell death. Collectively, in MG63 cells, AC exerted multiple effects on viability and [Ca²⁺]_i, caused apoptosis probably via inhibition of ERK MAPK phosphorylation. Drug Dev Res 68:71–78, 2007. © 2007 Wiley‐Liss, Inc.     

Validation of the intact rat weanling uterotrophic assay with notes on the formulation and analysis of the positive control chemical in vehicle

The intact female weanling version in the Organization for Economic Cooperation and Development (OECD) uterotrophic assay Test Guideline (TG) 440 is proposed as an alternative to the adult ovariectomized female version, because it does not involve surgical intervention (vs the ovariectomized version) and detects direct/indirect‐acting estrogenic/anti‐estrogenic substances (vs the ovariectomized version which detects only direct‐acting estrogenic/anti‐estrogenic substances binding to the estrogen receptor). This validation study followed OECD TG 440, with six female weanling rats (postnatal day 21) per dose group and six treatment groups. Females were weighed and dosed once daily by oral gavage for three consecutive days, with one of six doses of 17α‐ethinyl estradiol in corn oil at 5 ml kg^?1 at 0 and 0.1–10 µg kg^?1 per day. On postnatal day 24, the juvenile females were euthanized by CO₂ asphyxiation, weighed, livers weighed and uteri weighed wet and blotted. The presence or absence of vaginal patency was recorded. Absolute and relative (to terminal body weight) uterine wet and blotted weights and uterine luminal fluid weights were significantly increased at 3.0 and 10.0 (both P < 0.01) µg kg^?1 per day, and increased to ～140% of control values at 1.0 µg kg^?1 per day (not statistically significantly). In vivo body weights, weight changes, feed consumption, liver weights and terminal body weights were unaffected. Vaginal patency was not acquired in any female at any dose, although vaginal puckering was observed in one female at 10.0 µg kg^?1 per day. Therefore, this intact weanling uterotrophic assay is validated in our laboratory for use under US and European endocrine toxicity testing programs/legislation. Copyright © 2010 John Wiley & Sons, Ltd.     

A new Rhodamine-based turn-on fluorescent chemosensor for Fe3+

We have developed and characterized a new Rhodamine-based Fe³⁺ selective fluorescent turn-on chemosensor. A new Rhodamine-based fluorescent sensor Rh1 was synthesized by condensation and reduction of compound 1 with Salicylaldehyde. Fluorescent sensor Rh1 exhibited a good selectivity and little interference toward Fe³⁺ over other metal ions in acetonitrile. Fluorescent sensor Rh1 was colorless and non-fluorescent in the absence of Fe³⁺, pink color and strong fluorescence was observed after addition of Fe³⁺. Since the fluorescent sensor Rh1 undergoes 1,000 fold increase in fluorescence intensity along with color change upon binding with Fe³⁺, implying possible applications in a variety of area such as environmental monitoring and diag]nostic analysis.