Selenium deficiency in children and adolescents nourished by parenteral nutrition and/or selenium-deficient enteral formula

The authors analyzed serum selenium levels of 95 children and adolescents with intestinal dysfunction and/or neurological disabilities [age range: 7 months–20 years; mean ± standard deviation (SD): 8.0 ± 5.3 years] who received parenteral nutrition (PN) and/or enteral nutrition (EN) with either reduced or no selenium doses for more than 3 months. Twenty-eight patients (29%) showed serum selenium levels below 4.0 μg/dL. Five patients whose serum selenium levels were below 2 μg/dL presented various clinical manifestations, including hair browning (n = 5), macrocythemia (n = 4), nail whitening (n = 3) and cardiac dysfunction (n = 1). None of these 5 patients were nourished through ordinary diets. Three of these patients were nourished through selenium-free enteral nutritional products, 1 through selenium-deficient PN and 1 through PN and a formula with reduced selenium. After selenium supplement therapy for 1 year, all 5 patients exhibited improvement in their serum selenium levels and clinical features of selenium deficiency. It is important to be cautious about secondary selenium deficiency in children and adolescents nourished only through EN/PN without an adequate dose of selenium.     

Electrothermal atomic absorption spectrometry determination of aluminium in parenteral nutrition and its components

Aluminium concentration in samples of total parenteral nutrition solutions and samples of their individual components were analysed to know the exposure to this element. The median aluminium content obtained for the total parenteral nutrition solutions was 105.7 μg/L; for their individual components, 10% calcium gluconat and 1 M monopotasic phosphate were the most contaminated, as well as the 1 M sodium bicarbonate. The great variability found in the aluminium content of solutions suggests that contamination occurs during the manufacturing process.     

Brassica napus subjected to copper excess: Phospholipases C and D and glutathione system in signalling

Brassica napus plants were subjected to an oxidative stress by incubating them with 100 μM CuSO₄ for different times. The early response to copper stress was evaluated studying changes at both root and leaf level in the putative lipid and antioxidative signals diacylglicerol (DAG), phosphatidic acid (PA) and glutathione, in order to achieve elucidation on how these two kind of signals are related to each other. Activation of phospholipases C (PLC) and D (PLD) was studied in roots and leaves whereas increases in the levels of total and reduced glutathione (GSH) and changes in its redox status were evaluated in roots, leaves and chloroplast stroma. PLC and PLD were measured by studying the production of DAG, PA and phosphatidylbutanol (PtdButOH). PA, PtdButOH as well as DAG increased in roots already after 1 min of the treatment whereas in leaves, where no translocation of the metal occurred, any increase in PA and DAG was observed and no PtdButOH was formed. Roots were affected by oxidative stress showing decreases in glutathione reductase (GR), in total glutathione (GSH + GSSG) and GSH, and increases in oxidised glutathione (GSSG). In leaves, GR was induced during the whole stress period and both GSH + GSSG and GSH showed a peak at 5 min of the treatment. In the stroma, the maximum presence in GSH + GSSG and GSH occurred with a time shift of 25 min compared with total leaf extract.     

Effect of vitamin E and menadione supplementation on riboflavin production and stress parameters in Ashbya gossypii

Ashbya gossypii is a filamentous fungus which overproduces riboflavin as a pseudo-secondary metabolite. Vitamin E supplemented at 1, 2.5 and 5 μM levels in the growth medium of A. gossypii increased the extracellular secretion of riboflavin and at 50, 100 and 240 μM levels reduced the biomass and riboflavin yield. With 2.5 μM vitamin E total riboflavin production and extracellular riboflavin secretion on day 2 was higher than non-supplemented control. By day 3 the production in supplemented was nearly the same as in non-supplemented, but the intracellular riboflavin levels were lower and extracellular levels higher. Supplemented cells showed increased levels of catalase, glutathione peroxidase, lipid peroxides and membrane lipid peroxides, and decreased glutathione indicating that vitamin E, a well-known antioxidant, had acted as a pro-oxidant at low levels of 2.5 μM and had increased the oxidative stress. Menadione, a well known oxidant also increased riboflavin production and secretion at 1.0, 2.5 and 5.0 μM level. This is the first report were vitamin E and menadione effects support the concept that overproduction of riboflavin is a stress induced phenomenon. These findings are not only of scientific interest but also useful for improving the industrial production of riboflavin.     

HPLC analysis of human erythrocytic glutathione forms using OPA and N-acetyl-cysteine ethyl ester: Evidence for nitrite-induced GSH oxidation to GSSG

Glutathione exists in biological samples in the reduced form (GSH), as its disulfide (GSSG) and as a mixed disulfide (GSSR) with thiols (RSH). GSH is the most abundant low-molecular-mass thiol and plays important roles as a cofactor and as a main constituent of the intracellular redox status. Due to its own sulfhydryl (SH) group, GSH reacts readily with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative (GSH-OPA), which allows for sensitive and specific quantitative determination of GSH in biological systems by HPLC with fluorescence (FL) detection. In the present article we report on the utility of the novel, strongly disulfide bond-reducing thiol N-acetyl-cysteine ethyl ester (NACET) for the specific quantitative analysis of GSH and GSSG in the cytosol of red blood cells (RBC) as GSH-OPA derivative with FL (excitation/emission 338/458 nm) or UV absorbance (338 nm) detection. Unlike in aqueous solution, the derivatization of GSH in RBC cytosol yielded two closely related derivatives in the absence of NACET and only the GSH-OPA derivative in the presence of NACET. The HPLC method was optimized and validated for human RBC and applied to measure GSH and GSSG in RBC of healthy subjects. Basal GSH and GSSG concentrations were determined to be 2340 ± 350 μM and 11.4 ± 3.2 μM, respectively, in RBC of 12 healthy young volunteers (aged 23–38 years). The method was also applied to study the effects of nitrite on the glutathione status in intact and lysed human RBC. Nitrite at mM-concentrations caused instantaneous and considerable GSSG formation in lysed but much less pronounced in intact RBC. GSH externally added to lysed RBC inhibited nitrite-induced methemoglobin formation. Our findings suggest that nitric oxide/nitrite-related consumption rate of GSH, and presumably that of NADH and NADPH, could be of the order of 600 μmol/day in RBC of healthy subjects.     

Modulation of antioxidant enzyme activities and metabolites ratios by nitric oxide in short-term salt stressed soybean root nodules

Several abiotic factors cause molecular damage to plants either directly or through the accumulation of reactive oxygen species such as hydrogen peroxide (H₂O₂). We investigated if application of nitric oxide (NO) donor 2,2′-(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) could reduce the toxic effect resulting from short-term salt stress. Salt treatment (150 mM NaCl) alone and in combination with 10 μM DETA/NO or 10 μM DETA were given to matured soybean root nodules for 24 h. Salt stress resulted in high H₂O₂ level and lipid peroxidation while application of DETA/NO effectively reduced H₂O₂ level and prevented lipid peroxidation in the soybean root nodules. NO treatment increased the activities of ascorbate peroxidase and dehydroascorbate reductase under salt stress. Whereas short-term salt stress reduced AsA/DHAsA and GSH/GSSG ratios, application of the NO donor resulted in an increase of the reduced form of the antioxidant metabolites thus increasing the AsA/DHAsA and GSH/GSSG ratios. Our data suggests a protective role of NO against salt stress.     

Hemoglobin glutathionylation can occur through cysteine sulfenic acid intermediate: Electrospray ionization LTQ-Orbitrap hybrid mass spectrometry studies

Glutathionylated hemoglobin (Hb-SSG) is now recognized as a promising biomarker of systemic oxidative stress. Aim of this study is to gain a mechanistic insight into its formation. The ability of GSSG to form Hb-SSG through a thiol-disulfide exchange mechanism was firstly examined. For this purpose, GSSG (ranging from 0.23 to 230 μmol/g Hb, 15 μM–15 mM final concentrations) was incubated with 1 mM Hb and the relative content of Hb-SSG determined by direct infusion mass spectrometry (Orbitrap as analyzer). No detectable Hb-SSG was observed at a GSSG concentration range found in physiopathological conditions (0.13–0.23 μmol/g Hb). To reach a detectable Hb-SSG signal, the GSSG concentration was raised to 2.3 μmol/g Hb (0.5% relative abundance). The relative content of Hb-GSSG dose-dependently increased to 6% and 11% at 77 and 153 μmol/g Hb, respectively. The second step was to demonstrate whether Hb-SSG is formed through a sulfenic acid intermediate, a well-recognized mechanism of S-protein glutathionylation. Cys β93 sulfenic acid was found to be formed by oxidizing Hb with 1 mM H₂O₂, as demonstrated by direct infusion and LC–ESI-MS/MS experiments and using dimedone as derivatazing agent. When H₂O₂-treated Hb was incubated with physiological concentrations of GSH (9 μmol/g Hb), the corresponding Hb-SSG form was detected, reaching 15% of relative abundance. In summary, we here demonstrate that Hb glutathionylation can occur through a Cys sulfenic acid intermediate which is formed in oxidizing conditions. Hb glutathionylation is also mediated by a thiol-disulfide transfer mechanism, but this requires a concentration of GSSG which is far to be achieved in physiopathological conditions.     

In vitro plant regeneration,phenolic compound production and pharmacological activities of Coleonema pulchellum

Effects of plant growth regulators (PGRs) and organic elicitors (OEs) on Coleonema pulchellum in vitro micropropagation, secondary product production and pharmacological activities were evaluated. In vitro, ex vitro and parental plants of C. pulchellum were investigated for their potential to produce phenolic and pharmacological compounds. Different morphogenic characteristics of shoots were obtained with PGRs- and OEs-containing media. A higher number of normal shoots were achieved with a low concentration of thidiazuron (TDZ: 4.5 μM). Lesser numbers were found with combinations of TDZ (13.6 μM) + indole-3-acetic acid (IAA: 2.9 μM); haemoglobin (HB: 300 mg l^− 1) or glutamine (GM: 40 μM) + benzyladenine (BA: 8.8 μM). Shoots were rooted in vitro and successfully acclimatized. Plant growth regulators and OEs had a significant effect on the synthesis and accumulation of phenolic compounds and flavonoids. In particular, casein hydrolysate (CH) as well as a combination of GM and BA induced high levels of total phenolics and flavonoids during in vitro culture. Cytokinins and OEs had a significant effect on DPPH radical scavenging and antibacterial activities of C. pulchellum extracts. Acclimatized C. pulchellum plants can be used as substitute alternative to natural populations.     

Expression analysis and biochemical characterization of beans plants biofortificated with zinc

The present work was carried out in greenhouse conditions at the Centro de Investigación en Alimentación y Desarrollo AC in Delicias, Chihuahua, México. Four different concentrations (0, 25, 50 and 100 μM L⁻¹) of Zn chelate and sulfate were used to study the antioxidant system of Phaseolus vulgaris L. Three genes related with antioxidant activity [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] were selected for expression study. Results showed that when Zn chelate at 50 and 100 μM L⁻¹ were applied SOD was repressed and GSH-Px expression was low at 0, 25 and 100 μM L⁻¹ while with sulfate form SOD expression was low and GSH-Px expression was strong in all treatment. CAT was highly expressed in all form and treatments. For a biochemical study the same enzymes were spectrophotometrically measured. SOD activity shows differences in both forms of Zn, chelate form was different at 25, 50 and 100 μM L⁻¹ with less activity at 100 μM L⁻¹ and sulfate treatment shows differences in all concentrations used. GSH-Px activity shows significant differences with sulfate form at 25, 50 μM L⁻¹ where at 50 μM the activity was higher and low at 100 μM L⁻¹, CAT does not exhibit significant differences but with chelate treatment at 50–100 μM L⁻¹ the activity was higher compared to sulfate. Finally, to raise the Zn concentration in bean under biofortification program is a promising strategy in cropping systems in order to increase the ingestion of zinc and antioxidant capacity in the general population and provided the benefits that this element offered in human health.     

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

Alleviating the adverse effects of NaCl stress in maize seedlings by pretreating seeds with salicylic acid and 24-epibrassinolide

The effects of bio-regulators salicylic acid (SA) and 24-epibrassinolide (EBL) as seed soaking treatment on the growth traits, content of photosynthetic pigments, proline, relative water content (RWC), electrolyte leakage percent (EC%), antioxidative enzymes and leaf anatomy of Zea mays L. seedlings grown under 60 or 120 mM NaCl saline stress were studied. A greenhouse experiment was performed in a completely randomized design with nine treatments [control (treated with tap water); 60 mM NaCl; 120 mM NaCl; 10^− 4 M SA; 60 mM NaCl + 10^− 4 M SA; 120 mM NaCl + 10^− 4 M SA; 10 μM EBL; 60 mM NaCl + 10 μMEBL or 120 mM NaCl + 10 μM EBL] each with four replicates. The results indicated that NaCl stress significantly reduced plant growth traits, leaf photosynthetic pigment, soluble sugars, RWC%, and activities of catalase (CAT), peroxidase (POX) as well as leaf anatomy. However, the application of SA or EBL mitigated the toxic effects of NaCl stress on maize seedlings and considerably improved growth traits, photosynthetic pigments, proline, RWC%, CAT and POX enzyme activities as well as leaf anatomy. This study highlights the potential ameliorative effects of SA or EBL in mitigating the phytotoxicity of NaCl stress in seeds and growing seedlings.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Hepatocellular toxicity of clopidogrel: Mechanisms and risk factors

Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of this study was to find out the mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100 μM) was cytotoxic only after cytochrome P450 (CYP) induction by rifampicin. Clopidogrel (10 and 100 μM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100 μM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10 and 100 µM) in HepG2/CYP3A4, but not in HepG2/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10 and 100 µM) in HepG2/CYP3A4 supersome. Co-incubation with 1 μM ketoconazole or 10 mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 and HepG2/CYP3A4 supersome. HepG2/CYP3A4 incubated with 100 μM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100 µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites that are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.     

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (E_h) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 810±100 μmol/kg by 63 dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing E_h from −239±1 to −262±7 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and E_h were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.     

Thiol metabolism play significant role during cadmium detoxification by Ceratophyllum demersum L.

In the present study, the level of thiols and activity of related enzymes were investigated in coontail (Ceratophyllum demersum L.) plants to analyze their role in combating the stress caused upon exposure to cadmium (Cd; 0–10 μM) for a duration up to 7 d. Plants showed the maximum accumulation of 1293 μg Cd g^?1 dw after 7 d at 10 μM. Significant increases in the level of total non-protein thiols (NP-SH) including phytochelatins (PCs) as well as upstream metabolites of the PC biosynthetic pathway, cysteine and glutathione (GSH) were observed. In addition, significant increases in the activities of cysteine synthase (CS), glutathione-S-transferase (GST), glutathione reductase (GR), as well as in vitro activation of phytochelatin synthase (PCS), were noticed in response to Cd. In conclusion, under Cd stress, plants adapted to a new metabolic equilibrium of thiols through coordinated synthesis and consumption to combat Cd toxicity and to accumulate it.     

Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen is one of the main causes for the decline in motility and fertility of sperm during the freeze–thawing process. The aim of this study was thus to determine the effects of anti-oxidants on standard semen parameters, lipid peroxidation (LPO) and anti-oxidant activities after the freeze–thawing of ram semen. Ejaculates collected from four Akkaraman rams, were pooled and evaluated at 33 °C. Semen samples were diluted in a Tris-based extender containing the anti-oxidants glutathione (GSH) (5 mM), oxidized glutathione (GSSG) (5 mM) or cysteine (5 mM) and an extender containing no anti-oxidants (control), cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually for 20 s in a water bath (37 °C) for microscopic evaluation. The use of an extender supplemented with cysteine led to the highest (P < 0.01) post-thaw motility (61.0 ± 1.9%), compared to the other treatment groups. No significant differences were observed in viability, acrosome damage and total abnormalities, and following the hypo-osmotic swelling test (HOST), following supplementation with anti-oxidants after the thawing of the semen. Following the thawing process, the levels of malondialdehyde (MDA) did not change with the addition of anti-oxidants, compared to the control. The GSH level and glutathione peroxidase (GSH-PX) activity remained significantly higher upon the addition of GSH (3.33 ± 0.14 nmol/ml and 22.02 ± 1.27 IU/g protein) and GSSG (3.24 ± 0.08 nmol/ml and 20.17 ± 3.38 IU/g protein) compared to the other treatment (P < 0.001) groups. Only cysteine significantly elevated the activity of catalase (CAT, 842.40 ± 90.42 kU/l) following the freeze–thawing process. The Vitamin E (VitE) level was significantly higher, when compared to GSSG, cysteine and the control, when GSH (4.21 ± 0.20 mg/dl) was added to the freezing extender (P < 0.001). It could be concluded that future efforts aimed on improving the efficiency of cryopreservation of ram sperm should concentrate on the use of anti-oxidant additives. The results obtained provide a new approach to the cryopreservation of ram semen, and could positively contribute to intensive sheep production.     

Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.     

Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3 ± 1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7 μM IAA and 2.5 μM 2-isopentenyladenine (2iP) with 11.3 ± 0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.     

A simple glutathione transferase-based colorimetric endpoint assay for insecticide detection

The natural ability of the detoxification enzymes glutathione transferases (GSTs) to interact with xenobiotics can be used for the production of colorimetric assays. Detection is usually based on the inhibition of the GST-catalysed reaction, with detection achieved spectrophotometrically or electrochemically. Here we have adopted a chromogenic (visual) activity assay for screening GSTs with alkyltransferase activity for iodoalkene substrates for detection of insecticides. We screened a number of GSTs from insecticide resistant mosquito species for their ability to catalyse iodoalkane biotransformation reactions. AaGSTE2 was found to metabolise iodoethane with high turnover, which resulted in a dark blue colour in the enzymatic reaction. Following assay optimisation we exploited the high recognition affinity of the AgGSTE2 for insecticides to develop a novel colorimetric detection assay for organochlorine and pyrethroid quantification. Calibration curves were obtained for permethirn, deltamethrin, λ-cyhalothrin and DDT, with useful concentration ranges of 0–40 μg/ml (0–100 μM), 0–50 μg/ml (0–100 μM), 0–100 μg/ml (0–220 μM), and 0–50 μg/ml (0–140 μM), respectively. The assay was validated with extracts from insecticide sprayed surfaces and found to be reproducible and reliable compared with HPLC. The assay is therefore suitable for monitoring insecticide residues in insecticide treated materials, and therefore has potential for insect vector control operations.     

Identification of novel acetylcholinesterase inhibitors: Indolopyrazoline derivatives and molecular docking studies

The synthesis of novel indolopyrazoline derivatives (P1-P4 and Q1-Q4) has been characterized and evaluated as potential anti-Alzheimer agents through in vitro Acetylcholinesterase (AChE) inhibition and radical scavenging activity (antioxidant) studies. Specifically, Q3 shows AChE inhibition (IC₅₀: 0.68 ± 0.13 μM) with strong DPPH and ABTS radical scavenging activity (IC₅₀: 13.77 ± 0.25 μM and IC₅₀: 12.59 ± 0.21 μM), respectively. While P3 exhibited as the second most potent compound with AChE inhibition (IC₅₀: 0.74 ± 0.09 μM) and with DPPH and ABTS radical scavenging activity (IC₅₀: 13.52 ± 0.62 μM and IC₅₀: 13.13 ± 0.85 μM), respectively. Finally, molecular docking studies provided prospective evidence to identify key interactions between the active inhibitors and the AChE that furthermore led us to the identification of plausible binding mode of novel indolopyrazoline derivatives. Additionally, in-silico ADME prediction using QikProp shows that these derivatives fulfilled all the properties of CNS acting drugs. This study confirms the first time reporting of indolopyrazoline derivatives as potential anti-Alzheimer agents.