[Method for rapid synchronization of different growth cycles of Plasmodium falciparum in vitro and application in differential gene expression profile of 3D7 after dihydroartemisinin treatment].

Plasmodium culture in vitro is often used as an antimalarial drug evaluation model, but the lifecycle of P. falciparum culture in vitro tends to be disordered, which affects the research and evaluation of antimalarial drug mechanism in vitro. By combining magnetic bead separation method with sorbitol synchronization method, a synchronization method was constructed to quickly acquire different lifecycles of P. falciparum and obtain large amounts of parasite with a narrow synchronization window in a short period. Furthermore, the dihydroartemisinin(DHA) was used to treat the early trophozoite phase of P. falciparum 3 D7 for 4 h. Then mRNA was extracted and RNA-seq was conducted to analyze the differential expression of mRNA after drug treatment and obtain the differential gene expression profile. Differential expression of up-regulated genes and down-regulated genes was analyzed according to the screening criteria of |log_2FC|>1 and P<0.05. There, 262 genes were up-regulated and 77 genes were down-regulated. GO functional enrichment analysis of all the differentially expressed genes showed that the enrichment items mainly included cell membrane components, transporter activity, serine/threonine kinase activity, Maurer's clefts(MCs), rhoptry, antigen variation and immune evasion. The enrichment of KEGG pathway included malaria, fatty acid metabolism and peroxisome. Protein-protein interaction(PPI) analysis showed that the down-regulated genes in the modules with high degree of association included rhoptry, myosin complex, transporter and other genes related to the important life activities of malaria invasion and immune escape; the up-regulated genes were mainly related to various toxic exportins of malaria, such as PfSBP1 of MCs. qRT-PCR was used to verify the expression level of some genes, and most of the results were the same as the sequencing results. SBP1 was significantly up-regulated, while some antigenic protein expression levels were down-regulated. Above all, key molecules of DHA therapy were mainly involved in the parasites' rhoptry, transporter, antigenic variation, plasmodium exportin. These results offer us many hints to guide the further studies on mechanism of artemisinin and provide a new way for development of new antimalarial drugs.

Photoluminescence properties of a new orange-red-emitting Sm(3+)-La3SbO7 phosphor.

The antimonate compound La3SbO7 has high chemical stability, lattice stiffness and thermal stability. Orange-red-emitting antimonate-based phosphors La3SbO7:xSm(3+) (x = 0.02, 0.05, 0.08, 0.10, 0.15, 0.20 and 0.25) were synthesized. The phase structure and photoluminescence properties of these phosphors were investigated. The emission spectrum obtained on excitation at 407 nm contained exclusively the characteristic emissions of Sm(3+) at 568, 608, 654 and 716 nm, which correspond to the transitions from (4)G5/2 to (6)H5/2, (6)H7/2, (6)H9/2 and (6)H11/2 of Sm(3+), respectively. The strongest emission was located at 608 nm due to the (4)G5/2→(6)H7/2 transition of Sm(3+), generating bright orange-red light. The critical quenching concentration of Sm(3+) in La3SbO7:Sm(3+) phosphor was determined as 10% and the energy transfer between Sm(3+) was found to be through an exchange interaction. The International Commission on Illumination chromaticity coordinates of the La3SbO7:0.10Sm(3+) phosphors are located in the orange-red region. The La3SbO7:Sm(3+) phosphors may be potentially used as red phosphors for white light-emitting diodes.

[Assessment of Antimalarial Activity of Choline Derivatives against Plasmodium falciparum Growth in vitro by SYBR Green I Method].

OBJECTIVE: To investigate the antimalarial activity of four choline derivatives against Plasmodium falciparum 3D7 strain growth in vitro. METHODS: Four choline derivatives MD [N-dodecyl-N-(2-hydroxyethyl)-N,N- dimethyl ammonium bromide], ED [N-dodecyl-N-(2-hydroxyethyl)-N,N-diethyl ammonium bromide], MT [N-tetradecyl-N- (2-hydroxyethyl)-N,N-dimethyl ammonium bromide], and ET [N-tetradecyl-N-(2-hydroxyethyl)-N,N-diethyl ammonium bromide] were dissolved separately in DMSO at serial concentrations (1-10(5) µmol/L). The solutions were diluted by 1,000-fold with RPMI 1640 medium. 20 µl drug-containing medium and 80 µl P. falciparum-infected erythrocyte suspension (2% final hematocrit and 0.3%-0.5% parasitemia) were added to each well of microtiter plates. Drug effect on the in vitro growth of P. falciparum was measured by SYBR Green I method. The half maximal inhibitory concentration (IC50) was calculated from dose-response curves. Artemisinine served as positive control. RESULTS: Artemisinine, MD, ED, MT, and ET showed different degrees of dose-dependent inhibition on P. falciparum growth. When the MD concentration was above 10 nmol/L, the inhibition rate increased significantly. Both ED and ET showed significant inhibitory effects at high concentrations, with inhibition rate of > 95% when their doses were > 10(4) nmol/L. The IC50 values of MD, ED, MT, and ET were 1 620, 33.9, 116, and 68.9 nmol/L, respectively, all significantly higher than that of artemisinine (5.7 nmol/L) (P < 0.05). CONCLUSION: The four choline derivatives show certain antimalarial activity, which is lower than that of artemisinine. Among the four derivatives, ED has the strongest antimalarial activity against P. falciparum 3D7 strain.

Pharmacokinetics and tissue distribution of furanodiene W/O/W multiple emulsions in rats by a fast and sensitive HPLC-APCI-MS/MS method.

A sensitive and specific HPLC-APCI-MS/MS method was developed and validated for the quantification of furanodiene, a natural antitumor compound in rat plasma and tissues. W/O/W multiple emulsions of furanodiene, identified through microscope-observation and eosin staining method, were prepared with a two-step-procedure. Pharmacokinetics and tissue distribution were studied in rats after oral, intraperitoneal and intravenous injection with the dose of 5, 10 and 50 mg/kg, respectively. The assay achieved a good sensitivity and specificity for the determination of furanodiene in biological samples. The results showed that the concentration-time curves of furanodiene in rats after intravenous injection were fitted to a two-compartment model and the linear pharmacokinetic characteristic. The highest concentration in rat tissue was observed in the spleen, followed by heart, liver, lung, kidney, small intestine and brain. Comparing with the low concentration in plasma, furanodiene could be detected in various tissue samples after oral or intraperitoneal injection which indicated furanodiene had good and rapid tissue uptake. The results suggested that the wide tissue distribution of furanodiene could conduce to the therapeutic effects, but the short biological half-life limited its further application as an antitumor agent. The results are helpful for the structure modification of furanodiene as an antitumor candidate.

Xanthohumol Induces ROS through NADPH Oxidase, Causes Cell Cycle Arrest and Apoptosis.

Reactive oxygen species (ROS) are either toxic in excess or essential for redox signalling at the physiological level, which is closely related to the site of generation. Xanthohumol (XN) is an important natural product of hops (Humulus lupulus L.) and was reported to induce ROS in mitochondria. While in the present study, our data indicate that NADPH oxidase (NOX) is another site. In human acute myeloid leukemia HL-60 cells, we first identified that cell proliferation was inhibited by XN without affecting viability, and this could be alleviated by the antioxidant N-acetyl-L-cysteine (NAC); cell cycles were blocked at G1 phase, apoptosis was induced in a dose-dependent manner, and malondialdehyde (MDA) content was upregulated. XN-induced ROS generation was detected by flow cytometry, which can be inhibited by diphenyleneiodonium chloride (DPI, a NOX inhibitor), while not by NG-methyl-L-arginine acetate (L-NMMA, a nitric oxide synthase inhibitor). The involvement of NOX in XN-induced ROS generation was further evaluated: immunofluorescence assay indicated subunits assembled in the membrane, and gp91phox knockdown with siRNA decreased XN-induced ROS. Human red blood cells (with NOX, without mitochondria) were further selected as a cell model, and the XN-induced ROS and DPI inhibiting effects were found again. In conclusion, our results indicate that XN exhibits antiproliferation effects through ROS-related mechanisms, and NOX is a source of XN-induced ROS. As NOX-sourced ROS are critical for phagocytosis, our findings may contribute to the anti-infection and anti-inflammatory effect of XN.

Liquid chromatography-tandem mass spectrometry assay for the quantitation of beta-dihydroartemisinin in rat plasma.

Dihydroartemisinin (DHA) is a sesquiterpene used in the world as an antimalarial. To evaluate the pharmacokinetics of dihydroartemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantitation of dihydroartemisinin in rat plasma. For detection, a Sciex API 4000 LC-MS/MS with a TurboIonSpray ionization (ESI) inlet in the positive ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pre-treated by a simple liquid-liquid extraction with diethyl ether. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.2-100.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.2 ng/mL for beta-dihydroartemisinin in 100 microL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of beta-dihydroartemisinin in rats after oral administration.

[Simultaneous determination of metformin and glipizide in human plasma by liquid chromatography-tandem mass spectrometry].

To develop a sensitive and rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for simultaneous quantitation of metformin and glipizide in human plasma, metformin, glipizide and internal standard diphenhydramine were separated from plasma by protein precipitation with acetonitrile (containing 0.3% formic acid), then chromatographed by using a Zorbax Extend C18 column. The mobile phase consisted of acetonitrile-water-formic acid (70:30: 0.3, v/v/v), at a flow rate of 0.50 mL x min(-1). A tandem mass spectrometer equipped with atmospheric pressure chemical ionization source was used as detector and operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor/production combinations of m/z 130-->m/z 60, m/z 446-->m/z 321 and m/z 256-->m/z 167 were used to quantify metformin, glipizide and diphenhydramine, respectively. The linear concentration ranges of the calibration curves for metformin and glipizide were 2.00 - 2000 ng x mL(-1) and 1.00 - 1000 ng x mL(-1), respectively. The lower limits of quantitation of metformin and glipizide were 2.00 ng x mL(-1) and 1.00 ng x mL(-1), respectively. The method proved to be sensitive, simple and rapid, and suitable for clinical investigation of compound preparation containing metformin and glipizide.

[Metabolism of roxithromycin in dogs].

AIM: To investigate the metabolic profile of roxithromycin in dogs and the effects of oral and intravenous administrations on the metabolism of roxithromycin. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MSn) was used for separation and analysis of roxithromycin and its metabolites in dog bile after an oral dose or intravenous dose of roxithromycin. The metabolites were identified by comparisons of their mass spectra and LC behaviors with the references. RESULTS: Totally 13 metabolites were detected in dog bile, including N-demethylated derivatives, N, N-didemethylated derivatives, O-dealkylether derivatives, decladinose derivatives, and the geometric isomers of parent drug and its metabolites. CONCLUSION: Roxithromycin underwent 4 metabolic pathways in which geometric isomerization and decladinose metabolism were found to be markedly different between the two administration routes.

[Effects of wounding on jasmonic acid distribution in vicia faba leaves].

Higher plants have to cope with environmental stimulus such as wounding. Jasmonic acid (JA) is an essential long-distance signaling compound. There is rare information about JA cellular and subcellular localization by now. In this work, using the immuno-fluorescence and immuno-gold electron microscopy, distributions of JA were determined in different cells of Vicia faba leaf. It showed that JA existed in the epidermal cells, mesophyll cells and guard cells, mainly localized in the cytosol and chloroplast of mesophyll cells, cell wall of epidermal cells, and cytosol, cell wall, chloroplast and nucleus of guard cells. Wounding increased JA accumulation in the apoplast and guard cells. Our results suggest that JA plays an important role as a signal in the defense response and involves in regulation of the stomatal movement in response to wounding stress.

[Calcium involved in the signaling pathway of jasmonic acid induced stomatal closure of Vicia faba L].

Ca2+, an ubiquitous second messenger in the signal transudation pathway, is required for various physiological and developmental processes in plant. Jasmonic acid (JA) has been known to induce the stomatal closure. By monitoring the changes of [Ca2+]cyt with fluorescent probe Fluo-3 AM under the confocal microscopy, we observed that exogenous JA increased [Ca2+]cyt in guard cells of Vicia faba L. while the control and linolenic acid (LA), which is a precursor of JA, could hardly affect the change of [Ca2+]cyt. EGTA, a chelator of Ca2+ completely blocked JA-induced stomatal closure. After epidermis pretreated with EGTA, JA failed to result in [Ca2+]cyt increasing. Ruthenium red that blocked Ca2+ released from intracellular Ca2+ store could not significantly change JA-induced stomatal closure, while JA still increased [Ca2+]cyt. Furthermore, Ca2+ channel inhibitor of nifedipine (NIF) reduced the effectiveness of JA-induced stomatal closure and JA-induced increasing fluorescent intensity in guard cells. The results demonstrated that Ca2+ is involved in the signal transduction of JA induced stomatal closure, and the source of [Ca2+]cyt increasing in guard cells induced by JA might derive mainly from the external stores.

[The effect of gamma-aminobutyric acid in superoxide dismutase, peroxidase and catalase activity response to salt stress in maize seedling].

Salt stress induced Gamma-aminobutyric acid (GABA) accumulation in maize plants. The germination of maize seeds was inhibited seriously by NaCl treatment, while exogenous GABA reduced the inhibition of NaCl on the seeds germination. Effects on SOD, POD and CAT activity of GABA were detected. 1-2 mmol/L GABA induced the increase of the activity of SOD, POD and CAT about 20%. Because of SOD, CAT and POD are important protective enzymes which can eliminate active oxygen, so GABA can alleviate the damage of salt stress through promoting the activity of the protective enzyme system.

In vitro identification of metabolites of verapamil in rat liver microsomes.

AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 micromol/L and 5.0 micromol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MSn), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eight metabolites, including two novel metabolites (M4 and M8), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1-M4), N-dealkylated derivatives of verapamil (M5-M7), and N,O-didemethyl-verapamil (M8). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.

[Redistribution of cell contents between declining old and growing young leaves in spring onion].

During storage of a spring onion (Allium fistulosa var. dacong), the plant is unable to assimilate external resource, nutrient requirement of the new bud is entirely drawn from the withering leaf where both food reserves and protoplasmic constituents (i.e. organic complexes containing the essential mineral elements) are to be mobilized. Evidences were given to show that the withdrawal process of cell contents from the old part for establishing the new growth should be considered as an alternative mode or a supplementary measure of normal phloem transport of leaf photosynthates. Removal of cell contents from the declining old part to the developing new growth is of frequent occurrence in higher plants. In the present case, it followed the process of programmed cell death (PCD). That was examined and shown by electron microscopy and biochemical analysis. There appeared shrinkage of cell volume, dissolution of cell constitution and degradation of DNA. The present investigation has also shown that the withdrawal of cell contents from the declining leaf sheath of spring onion can be wholly exhaustive. It may be carried out by active movement of the partially degraded protoplasmic constituents in forms of fragments and vesicles across the tissue, until hardly anything in the cellulose framework of the dry papery sheath, except a few crystals of Ca2+. Presence of high ATPase and APase activities along the transport path also supports this view.

Metabolism of roxithromycin in phenobarbital-treated rat liver microsomes.

AIM: To investigate the metabolism of roxithromycin (RXM) in rat liver microsomes and the possible effects of RXM and its metabolites on cytochrome P-450 (CYP450). METHODS: Liver microsomes of Wistar rats, induced by phenobarbital, were prepared using ultracentrifuge method. RXM in vitro metabolism was stu died with the microsome incubation. The metabolites were separated and assayed by li quid chromatography-tandem mass spectrometry (LC-MSn), and were further identified by comparison of their mass spectra and LC behavior to synthesized references. RESULTS: N-Mono- and N-di-demethyl metabolites a s well as O-dealkylated metabolite (erythromycin oxime) were detected in microsomal incubates. RXM and its metabolites expressed weak potency to form inactive complexes with CYP450. CONCLUSION: N-Demethylation and oxime ether side chain O- dealkylation are main biotransformation pathways of RXM in phenobarbital-treated rat liver microsomes. Both routes were found to be NADPH-dependent. RXM and its metabolites showed weak inhibitory effects on CYP450.