AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6.

Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two Orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, Oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three Pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.

Identification and functional characterization of a PDR transporter in Tripterygium wilfordii Hook.f. that mediates the efflux of triptolide.

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.

A class I TGA transcription factor from Tripterygium wilfordii Hook.f. modulates the biosynthesis of secondary metabolites in both native and heterologous hosts.

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.

[Inhibition of MCF-7/ADR cells by DOX-loaded pluronic-attached PAMAM dendrimer conjugate].

Pluronic modified polyamidoamine (PAMAM) conjugate (PF127-PAMAM) was prepared and the inhibiting effect of MDR against MCF-7/ADR was investigated with doxorubicin (DOX) as model drug. 1H NMR and FTIR spectra showed that the conjugate was synthesized successfully. Element analysis accurately measured that 27.63% amino of per PAMAM was modified by pluronic (PAMAM : PF127, 1 : 35.37 mole ratio). PF127-PAMAM showed an increased size and a reduced zeta potential compared to PAMAM. PF127-PAMAM had lower hemolytic toxicity and cytotoxicity due to the reduced zeta potential and the protection of PF127. Each PF127-PAMAM molecular could load 19.58 DOX molecules, and the complex exhibited sustained and pH-sensitive release behavior. PF127-PAMAM/DOX exhibited weaker cytotoxicity than free DOX in MCF-7 cells; while the complex showed much stronger reverse effect of drug resistance in MCF-7/ADR cells, and resistance reversion index (RRI) was as high as 33.15.

[Analysis and assessment of atmospheric pollution based on accumulation characterization of heavy metals in Platanus acerifolia leaves].

The present work was aimed to evaluate the heavy metal pollution in the atmosphere of Huainan City. We measured and clustered the accumulation of six heavy metals in Platanus acerifolia leaves in 20 sampling fields with six types of environmental conditions, and analyzed the EF value of heavy metal enrichment in the leaves. The results showed that the accumulations in Platanus acerifolia leaves varied according to different types of metals, following the order of Zn > Cu > Cr > Ni > Pb > Cd. Environmental conditions also had great influence on the accumulation of heavy metals. Cd and Cu were mostly found in cement plant and mine, respectively, and Cr, Ni, Pb and Zn were significant higher in main road, compared with other environmental conditions. The average values of EF for all the metals expect Cr in scenic and village area were over 1. The average values of EF for all the metals in mine, power plant, main road and cement plant were above 3. The overall pollution condition of heavy metals in Huainan City followed the order of Cd > Cu > Zn > Ni > Pb > Cr.

Lanthanum ions intervened in enzymatic production and elimination of reactive oxygen species in leaves of rice seedlings under cadmium stress.

Rare earth elements at proper concentrations were observed to alleviate heavy metal stress. However, the potential mechanisms of alleviation are not clear. Thus, the present study focused on the mechanisms of lanthanum (La) ions on the generation and elimination of reactive oxygen species (ROS) production in leaves of rice seedlings hydroponically cultivated in 30 µM cadmium chloride (CdCl2) for 2 d. Results showed that the decline of superoxide (O2 · (-)) or hydrogen peroxide (H2O2) was attributed not only to the decreased expressions of nicotinamide adenine dinucleotide phosphate oxidases-related gene, protein synthesis, and isozymes, but also to the decrease of xanthine oxidase, diamine oxidase, and guaiacol peroxidases isozymes and activities. Genes and isozymes related to superoxide dismutase, catalase, and ascorbate peroxidase were also differentially upregulated to control ROS accumulation when proper concentrations of La were combined with 30 µM Cd. Based on the results, one may conclude that La at proper concentrations not only limited the generation of O2 · (-) and H2O2 from the enzymatic sources but also promoted the elimination of O2 · (-) and H2O2 via the upregulating expressions of antioxidant enzyme-related genes and activities in the rice seedling's leaves under Cd stress.

[Preparation and in vitro evaluation of doxorubicin-loaded magnetic iron oxide nanoparticles].

PEG-modified magnetic Fe3O4 (Fe3O4-PEG) nanoparticles were sythesized using a solvothermal reaction and characterized with transmission electron microscopy (TEM) and thermo gravimetric analysis (TGA). The photothermal effect and photothermal destruction of cancer cells were evaluated. Then the doxorubicin loaded Fe3O4-PEG (DOX-Fe3O4-PEG) nanoparticles were prepared. The cytotoxicity and combined chemotherapy/photothermal therapy (PTT) effect were investigated. Uniform PEG coated Fe3O4 nanoparticles with particle size of 155 nm were obtained in the experiment. The loading and release of doxorubicin on Fe3O4-PEG were pH-dependent. The drug loading capacity in water was 21%. The results of MTT indicated a good biocompatiblity of Fe3O4-PEG nanoparticles and high cytotoxicity of DOX-Fe3O4-PEG. In combined therapy experiment, photothermal therapy demonstrated unambiguously enhanced chemotherapy efficacy. In conclusion, the obtained Fe3O4-PEG nanoparticles which exhibit good photothermal effect and drug loading capacity can be used for chemotherapy and photothermal therapy. The synergetic anti-tumor activity indicates the potential for the combined application of chemotherapy and photothermal therapy in cancer treatment.

Antioxidant and prooxidant effects of lanthanum ions on Vicia faba L. seedlings under cadmium stress, suggesting ecological risk.

The present study combined chemical analyses and biological measurements to investigate biphasic effects of La on Cd stress in leaves of Vicia faba seedlings, which were hydroponically cultivated for 15 d in the combination of 6 µM CdCl(2) and 2 to 480 µM La(NO(3))(3), respectively. The results showed that contents of Cd first elevated above and then declined below the 6 µM single Cd treatment when 2 to 30 µM extraneous La were combined. Contents of mineral nutrients altered differentially and became imbalanced. No distinct band was observed in catalase (CAT), guaiacol peroxidase (GPX), or ascorbate peroxidase (APX) patterns, but in superoxide dismutase (SOD) isozymes by the supplementation with 8 to 480 µM of extraneous La. Superoxide dismutase and APX activities changed as a U-shaped curve; however, CAT and GPX changed as an inverted U-shaped curve along with increasing La. Moreover, heat shock protein 70 (HSP 70) production was reduced below the single treatment of Cd at 2 to 8 µM of extraneous La and enhanced thereafter. Thus, La at lower concentrations promoted antioxidation against Cd stress; La at higher concentrations turned to prooxidant effects, implicating potential ecological risk. Heat shock protein 70, combined with the antioxidant enzymes, constitutes an integrative defense system, which can be used to estimate the degree of antioxidation or prooxidation of extraneous La to Cd-induced oxidative stress in the seedlings.

[Preparation of pantoprazole sodium enteric-coated pellets-type tablets].

This study is to prepare the pantoprazole sodium enteric-coated tablet which is compacted by pellets. The enteric-coated pantoprazole sodium pellets were prepared by fluid bed coating technology. The pantoprazole sodium enteric-coated tablets were prepared by direct compression of the enteric-coated pellets and suitable excipients. In vitro dissolution method and scanning electron microscope method were used for the observation of the drug release behavior before and after compression of the pellets. The optimized formulation is: the coating level is 55%, the plasticizer content is 20%, the ratio of Eudragit L30D-55/NE30D is 8 : 2, enteric-coated pellets/excipients (MCC/PPVP/PEG 6000 = 2 : 1 : 1) is 5 : 5, the enteric-coated tablets release in artificial gastric fluid in 2 h is less than 10%, while in artificial intestinal fluid in 1 h is more than 85%. The release behavior of pantoprazole sodium enteric-coated pellets-type tablet is quite well. And it may be used in industrial production.

[Preparation and evaluation of enteric-coated and taste masking clarithromycin granules].

The study is to prepare taste masking and enteric-coated clarithromycin granules by melting and fluid bed coating technology. Clarithromycin and matrix materials were melted at a certain temperature, and then made into particles by fluidized bed coating. X-ray powder diffraction and scanning electron microscopy were used to identify the crystal and morphology of drug loading granules. In vitro dissolution method was used for the observation of the drug release behavior. The results showed that the drug particles size range was 0.2 - 0.6 mm; the crystal form of clarithromycin in the granule did not change; enteric-coated granules accumulated release in 0.1 mol L(-1) hydrochloric acid in 2 h was less than 10%, while in pH 6.8 phosphate buffer in 1 h was more than 80%. The taste masking and enteric-coated clarithromycin granules not only have good taste masking effect, but also have a good release behavior. It is expected to have better clinical application.

Hormesis effects and implicative application in assessment of lead-contaminated soils in roots of Vicia faba seedlings.

Chemical analyses and biological methods were combined to investigate oxidative stress, hormesis effect and concerned mechanism in roots of Viciafaba seedlings grown in 0-2000 mg kg(-1) of Pb-treated soils after germination of 20d. The results showed that U-shaped dose response curves were displayed in superoxide radical (O2-) radicals, guaiacol peroxidase (POD) and ascorbate peroxidase (APX) activities, malondialdehyde (MDA) and carbonyl groups as well as activities of endoproteinase (EP) isoenzymes in the roots at low doses of extraneous Pb, indicating reduced oxidative stress and toxic effect. The inverted U-shaped curves were also exhibited in growth height, superoxide dismutase (SOD) and EP activities as well as inducible heat shock protein70 (HSP70) with the increasing extraneous Pb, indicative of enhanced oxidative stress. The enhancement in HSP70, carbonyl groups and EP activities confirmed intracellular proteotoxicity and proteolytic activity in the roots at higher doses of soil Pb. More interestingly, levels of inducible HSP70 were well correlated with those of growth heights (r=0.809, p<0.05), implying that HSP70 induction may be one of the mechanisms underlying the U-shaped growth curve of V. faba seedlings in the experiment. The results suggest that traditional threshold models ought to be combined with hormesis effect in assessment of Pb-polluted soils and the threshold dose range of Pb-treated soils is proposed rudimentally as 25-125 mg kg(-1).

Oxidative stress, defense response, and early biomarkers for lead-contaminated soil in Vicia faba seedlings.

Chemical analyses and biological measurements were investigated in leaves of Vicia faba seedlings exposed to extraneous lead (Pb) at 0 to 2,000 mg/kg of soil for a month. The results showed that superoxide radical (O*(2)(-)) production, increased along with total Pb in leaves and available Pb in soil, resulted in enhancement of malondialdehyde and carbonyl groups. Antioxidant enzymes, including corresponding isoenzymes and heat shock protein 70 (hsp 70), were also enhanced to some extent. Significant changes were detected in the patterns and intensities of guaiacol peroxidase isoenzymes, while superoxide dismutase, catalase, and ascorbate peroxidase isoenzymes only changed intensities. Superoxide dismutase activities increased with the increase of extraneous Pb at 0 to 500 mg/kg of soil and tended to decline thereafter, which might be responsible for the decrease of hydrogen peroxide and accumulation of O*(2)(-). Guaiacol peroxidase and ascorbate peroxidase enzymes were upregulated to become major scavengers of excess hydrogen peroxide on the condition of decreased catalase activities. Levels of hsp 70 were well correlated with Pb contents in leaves (r=0.777), O*(2)(-) accumulation (r = 0.985, p<0.01), and carbonyl groups (r=0.920, p<0.01) under extraneous Pb at 0 to 250 mg/kg of soil, suggesting that hsp 70 induced by O*(2)(-) was possibly involved in disposal of denatured proteins. The results showed that O*(2)(-), hsp 70, and guaiacol peroxidase isoenzymes had the most sensitive responses in the seedlings and these parameters could be potential early biomarkers of soil Pb contamination.

[Evaluation of soil lead contamination by biomarkers in leaves of Vicia faba L. seedlings].

Stress proteins (HSP70 and HSP60) and other related biological parameters were investigated in leaves of Vicia faba L. seedlings subjected to soil lead stress. The seedlings were cultivated outdoors in pots for five weeks. Results showed that total lead content, superoxide radical (O2*-) and lipid peroxidation productions in the leaves increased with the increase of extraneous lead in soils. Significant correlation was found between the O2*- and lipid peroxidation productions (r = 0.973, p < 0.01). Activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were activated. Moreover, HSP70 and HSP60 increased more sensitively than the other parameters in response to lead stress. This indicates lead might cause lipid peroxidation in the leaves by mediation of O2*-. The activated antioxidative enzymes alleviated the stress and damage of reactive oxygen species. HSP70 and HSP60 are potential biomarkers for early warning of lead-contaminated soils. Additionally, concentrations of soil lead and related biological parameters should be combined.

[Research on the cytotoxic and genotoxic effects of rare-earth element holmium to Vicia faba].

Crystal of nitrate, made by the reaction of holmium trioxide and nitric acid, was dissolved in distilled water, thus diluted into gradient solution. Soaked in the solution for 6 hours (6h), the root tips of Vicia faba were then recovered and cultivated for 22 h and 24 h, respectively. By observing the change of root tips and calculating the frequency of micronucleus (FMN), the frequency of chromosomal aberrations(CAF) and mitosis index (MI),we find that the dosage below 4mg/L (expressed by concentration of holmium trioxide) could accelerate the growth of root tips of Vicia faba. CAF and FMN increased while MI decreased with the rise of concentrations. From it a dosage effect relationship is clearly seen. And it indicated that the rare earth element holmium has certain cytotoxic and genotoxic effects. Furthermore, the different recovery groups have different FMN, CAF and MI, and the difference lies in the fact that FMN of 22 h recovery group was lower than that of 24 h recovery group, while CAF and MI were higher than those of 24 h recovery group. The results suggest that the statistics of FMN should be made after that of CAF.

Investigations on factors that influence the moving neutralization reaction boundary method for capillary electrophoresis and isoelectric focusing.

We investigated several factors, such as temperature, current intensity (i), time (t) and the product (mA min mm(-2), viz., C mm(-2)) of i and t, etc., that obviously affect the moving neutralization reaction boundary method (MNRBM). The results manifest that the temperature and the product ti have a strong influence on the movement rate of the boundary. The data prove that about 0.6 C mm(-2) (being equivalent to 10 mA min mm(-2)) is a critical point. If the product ti is lower than the critical point, a good quantitative agreement exists between the observed and theoretical values, but if it is higher than the critical point, the agreements are poor. The optimized experimental conditions are: (1) 18-20 degrees C room temperature, (2) 0.6-0.8 mA mm(-2), (3) less than 10 mA min mm(-2), (4) 1% agarose gel, (5) daily prepared solution and gel containing NaOH. The optimized MNRBM is of benefit for the studies on MNRB itself, isoelectric focusing and capillary zone electrophoresis as will be partially shown in this paper.