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1.  Effects of Ginkgo biloba Extract on Inflammatory Mediators (SOD, MDA, TNF-α, NF-κBp65, IL-6) in TNBS-Induced Colitis in Rats 
Mediators of Inflammation  2006;2006(5):92642.
Inflammatory mediators play a criticial role in ulcerative colitis immune and inflammatory processes. The aim of the study was to investigate the effects of Ginkgo biloba extract on inflammatory mediators (SOD, MDA, TNF-α, NF-κBp65, IL-6) in TNBS-induced colitis in rats. Colitis in rats was induced by colonic administration with 2,4,6-trinitrobenzene sulfonic acid (TNBS, 150 mg/kg). EGB in doses of (50, 100, 200 mg/kg) was administered for 4 weeks to protect colitis. The results showed that EGB could significantly ameliorate macroscopic and histological damage, evidently elevate the activities of SOD and reduce the contents of MDA, inhibit the protein and mRNA expressions of TNF-α, NF-κBp65, and IL-6 in the colon tissues of experimental colitis in a dose-dependent manner compared with the model group. We concluded that the probable mechanisms of EGB ameliorated inflammatory injury in TNBS-induced colitis in rats by its modulation of inflammatory mediators and antioxidation
PMCID: PMC1657076  PMID: 17392580
2.  Effects of silencing RIP1 with siRNA on the biological behavior of the LoVo human colon cancer cell line 
Oncology Letters  2014;7(6):2065-2072.
The present study aimed to investigate the effects of silencing RIP1 by small interfering RNA (siRNA) on the biological behavior of the LoVo human colorectal carcinoma cell line and to provide evidence for the feasibility of colorectal cancer gene therapy. LoVo cells were divided into the RIP1 siRNA group, the blank control group and the negative control group. Chemically synthesized siRNA targeting RIP1 (RIP1 siRNA) was transfected into LoVo cells. Following transfection of the RIP1-targeted siRNA into the LoVo cells, the expression of the RIP1 gene was effectively inhibited. The results demonstrated that RIP1 effectively regulated the malignant biological behavior of the LoVo colon cancer cell line. Furthermore, the proliferation, motility and invasiveness of LoVo cells were inhibited by siRNA knockdown of RIP1. The results revealed that the RIP1 gene has an important role in the regulation of proliferation and apoptosis in colorectal carcinoma cells.
PMCID: PMC4049674  PMID: 24932290
RNA interference; RIP1 gene; colorectal carcinoma
3.  Effect and mechanism of the Twist gene on invasion and metastasis of gastric carcinoma cells 
AIM: To study the effect of the transfected Twist gene on invasion and metastasis of gastric carcinoma cells and the possible mechanisms involved.
METHODS: Human gastric carcinoma MKN28 cells were stably transfected with Twist sense plasmid, and MKN45 cells were stably transfected with Twist antisense plasmid using the lipofectamine transfection technique. RT-PCR, Western blotting, EMSA, gelatin zymography assay, and in vitro invasion and migration assays were performed. Nude mice metastasis models were established by the abdominal cavity transfer method.
RESULTS: Cell models (TwistS-MKN28) that steadily expressed high Twist protein were obtained. Compared with MKN28 and pcDNA3-MKN28 cells, adherence, migration and invasion ability of TwistS-MKN28 cells were clearly raised. The number of cancer nodules was increased significantly in the abdominal cavity and liver of nude mice inoculated with TwistS-MKN28 cells. Overexpression of Twist in MKN28 cells increased Tcf-4/Lef DNA binding activity, and promoted expression of Tcf-4’s downstream target genes cyclin D1 and MMP-2. However, suppression of Twist (TwistAS-MKN45) inhibited MKN45 cell invasion and the expression of cyclin D1 was reduced. The activity of MMP-2 was also decreased.
CONCLUSION: These results indicate that Twist promotes gastric cancer cell migration, invasion and metastasis, and Twist may play an important role in Wnt/Tcf-4 signaling.
PMCID: PMC2708358  PMID: 18442194
Twist; Gastric carcinoma; Cancer invasion; Metastasis; Tcf4
4.  Effect of ligand troglitazone on peroxisome proliferator-activated receptor γ expression and cellular growth in human colon cancer cells 
AIM: To investigate the effect of troglitazone on pe-roxisome proliferator-activated receptor γ (PPARγ) expression and cellular growth in human colon cancer HCT-116 and HCT-15 cells and to explore the related molecular mechanism.
METHODS: Human colon cancer HCT-116 and HCT-15 cells cultured in vitro were treated with troglitazone. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were employed to detect the effect of troglitazone on PPARγ expression. The proliferative activity was determined by MTT assay, cell cycle and apoptosis were detected by flow cytometry. Apoptosis-related genes, cell cycle regulatory genes and p53 were examined by RT-PCR and Western blot respectively.
RESULTS: The expression of PPARγ in colon cancer HCT-116 and HCT-15 cells was up-regulated by troglitazone. Troglitazone inhibited proliferation, induced apoptosis and cell cycle G1 arrest in colon cancer cells. Troglitazone induced p53 expression in HCT-116 cells, but not in HCT-15 cells. The down-regulation of survivin and bcl-2 was found in both cell lines and up-regulation of bax was found only in HCT-116 cells, being consistent with growth inhibition in HCT-116 cells but not in HCT-15 cells. Troglitazone increased expression of p21WAF1/CIP1 (p21), p27KIP1 (p27) and reduced cyclin D1 in HCT-116 cells while only a minor decrease of cyclin D1 was found in HCT-15 cells.
CONCLUSION: Troglitazone is an inductor of PPARγ in colon cancer cells and inhibits PPARγ-dependently proliferation, which may attribute to cell cycle G1 arrest and apoptosis in colon cancer cells. Troglitazone may induce p53-independent apoptosis and p53-dependent expression of p21 and p27. Depending on cell background, different activation pathways may exist in colon cancer cells.
PMCID: PMC4087481  PMID: 17143939
Peroxisome proliferator-activated receptor γ; Colon cancer; Troglitazone; Cellular growth; Cell cycle; Apoptosis
5.  H2O2 mediates the crosstalk of brassinosteroid and abscisic acid in tomato responses to heat and oxidative stresses 
Journal of Experimental Botany  2014;65(15):4371-4383.
Brassinosteroids induce H2O2 accumulation from RBOH1-NADPH oxidase, which first induces ABA biosynthesis and stress tolerance, in turn leading to prolonged H2O2 production in both apoplast and chloroplast and stress tolerances.
The production of H2O2 is critical for brassinosteroid (BR)- and abscisic acid (ABA)-induced stress tolerance in plants. In this study, the relationship between BR and ABA in the induction of H2O2 production and their roles in response to heat and paraquat (PQ) oxidative stresses were studied in tomato. Both BR and ABA induced increases in RBOH1 gene expression, NADPH oxidase activity, apoplastic H2O2 accumulation, and heat and PQ stress tolerance in wild-type plants. BR could only induced transient increases in these responses in the ABA biosynthetic mutant notabilis (not), whereas ABA induced strong and prolonged increases in these responses in the BR biosynthetic mutant d ^im compared with wild-type plants. ABA levels were reduced in the BR biosynthetic mutant but could be elevated by exogenous BR. Silencing of RBOH1 compromised BR-induced apoplastic H2O2 production, ABA accumulation, and PQ stress responses; however, ABA-induced PQ stress responses were largely unchanged in the RBOH1-silenced plants. BR induces stress tolerance involving a positive feedback mechanism in which BR induces a rapid and transient H2O2 production by NADPH oxidase. The process in turn triggers increased ABA biosynthesis, leading to further increases in H2O2 production and prolonged stress tolerance. ABA induces H2O2 production in both the apoplastic and chloroplastic compartments.
PMCID: PMC4112640  PMID: 24899077
Abscisic acid; brassinosteroid; hydrogen peroxide; NADPH oxidase; Solanum lycopersicum; VIGS.
6.  Chloroplastic thioredoxin-f and thioredoxin-m1/4 play important roles in brassinosteroids-induced changes in CO2 assimilation and cellular redox homeostasis in tomato 
Journal of Experimental Botany  2014;65(15):4335-4347.
Virus-induced gene silencing (VIGS) was used in this study to characterize the role of thioredoxin-f and thioredoxin-m1/4 in brassinosteroid-induced changes in CO2 assimilation and cellular redox homeostasis in tomato.
Chloroplast thioredoxins (TRXs) and glutathione function as redox messengers in the regulation of photosynthesis. In this work, the roles of chloroplast TRXs in brassinosteroids (BRs)-induced changes in cellular redox homeostasis and CO2 assimilation were studied in the leaves of tomato plants. BRs-deficient d ^im plants showed decreased transcripts of TRX-f, TRX-m2, TRX-m1/4, and TRX-x, while exogenous BRs significantly induced CO2 assimilation and the expression of TRX-f, TRX-m2, TRX-m1/4, and TRX-x. Virus-induced gene silencing (VIGS) of the chloroplast TRX-f, TRX-m2, TRX-m1/4, and TRX-y genes individually increased membrane lipid peroxidation and accumulation of 2-Cys peroxiredoxin dimers, and decreased the activities of the ascorbate–glutathione cycle enzymes and the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in the leaves. Furthermore, partial silencing of TRX-f, TRX-m2, TRX-m1/4, and TRX-y resulted in decreased expression of genes involved in the Benson–Calvin cycle and decreased activity of the associated enzymes. Importantly, the BRs-induced increase in CO2 assimilation and the increased expression and activities of antioxidant- and photosynthesis-related genes and enzymes were compromised in the partially TRX-f- and TRX-m1/4-silenced plants. All of these results suggest that TRX-f and TRX-m1/4 are involved in the BRs-induced changes in CO2 assimilation and cellular redox homeostasis in tomato.
PMCID: PMC4112637  PMID: 24847092
Antioxidant; Benson–Calvin cycle; chloroplast; 2-Cys peroxiredoxin; glutathione; photosynthesis.
7.  RBOH1-dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation-induced cross-tolerance in tomato 
Journal of Experimental Botany  2013;65(2):595-607.
H2O2 and mitogen-activated protein kinase (MAPK) cascades play important functions in plant stress responses, but their roles in acclimation response remain unclear. This study examined the functions of H2O2 and MPK1/2 in acclimation-induced cross-tolerance in tomato plants. Mild cold, paraquat, and drought as acclimation stimuli enhanced tolerance to more severe subsequent chilling, photooxidative, and drought stresses. Acclimation-induced cross-tolerance was associated with increased transcript levels of RBOH1 and stress- and defence-related genes, elevated apoplastic H2O2 accumulation, increased activity of NADPH oxidase and antioxidant enzymes, reduced glutathione redox state, and activation of MPK1/2 in tomato. Virus-induced gene silencing of RBOH1, MPK1, and MPK2 or MPK1/2 all compromised acclimation-induced cross-tolerance and associated stress responses. Taken together, these results strongly suggest that acclimation-induced cross-tolerance is largely attributed to RBOH1-dependent H2O2 production at the apoplast, which may subsequently activate MPK1/2 to induce stress responses.
PMCID: PMC3904713  PMID: 24323505
Cross-tolerance; hydrogen peroxide; mitogen-activated protein kinase; reactive oxygen species; Respiratory burst oxidase homologue 1; signal transduction; Solanum lycopersicum.
8.  The Role of Hydrogen Peroxide and Nitric Oxide in the Induction of Plant-Encoded RNA-Dependent RNA Polymerase 1 in the Basal Defense against Tobacco Mosaic Virus 
PLoS ONE  2013;8(9):e76090.
Plant RNA-dependent RNA Polymerase 1 (RDR1) is an important element of the RNA silencing pathway in the plant defense against viruses. RDR1 expression can be elicited by viral infection and salicylic acid (SA), but the mechanisms of signaling during this process remains undefined. The involvement of hydrogen peroxide (H2O2) and nitric oxide (NO) in RDR1 induction in the compatible interactions between Tobacco mosaic tobamovirus (TMV) and Nicotiana tabacum, Nicotiana benthamiana, and Arabidopsis thaliana was examined. TMV inoculation onto the lower leaves of N. tabacum induced the rapid accumulation of H2O2 and NO followed by the increased accumulation of RDR1 transcripts in the non-inoculated upper leaves. Pretreatment with exogenous H2O2 and NO on upper leaf led to increased RDR1 expression and systemic TMV resistance. Conversely, dimethylthiourea (an H2O2 scavenger) and 2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (an NO scavenger) partly blocked TMV- and SA-induced RDR1 expression and increased TMV susceptibility, whereas pretreatment with exogenous H2O2 and NO failed to diminish TMV infection in N. benthamiana plants with naturally occurring RDR1 loss-of-function. Furthermore, in N. tabacum and A. thaliana, TMV-induced H2O2 accumulation was NO-dependent, whereas NO generation was not affected by H2O2. These results suggest that, in response to TMV infection, H2O2 acts downstream of NO to mediate induction of RDR1, which plays a critical role in strengthening RNA silencing to restrict systemic viral infection.
PMCID: PMC3786905  PMID: 24098767
9.  Hydrogen peroxide functions as a secondary messenger for brassinosteroids-induced CO2 assimilation and carbohydrate metabolism in Cucumis sativus *  
Brassinosteroids (BRs) are potent regulators of photosynthesis and crop yield in agricultural crops; however, the mechanism by which BRs increase photosynthesis is not fully understood. Here, we show that foliar application of 24-epibrassinolide (EBR) resulted in increases in CO2 assimilation, hydrogen peroxide (H2O2) accumulation, and leaf area in cucumber. H2O2 treatment induced increases in CO2 assimilation whilst inhibition of the H2O2 accumulation by its generation inhibitor or scavenger completely abolished EBR-induced CO2 assimilation. Increases of light harvesting due to larger leaf areas in EBR- and H2O2-treated plants were accompanied by increases in the photochemical efficiency of photosystem II (ΦPSII) and photochemical quenching coefficient (q P). EBR and H2O2 both activated carboxylation efficiency of ribulose-1,5-bisphosphate oxygenase/carboxylase (Rubisco) from analysis of CO2 response curve and in vitro measurement of Rubisco activities. Moreover, EBR and H2O2 increased contents of total soluble sugar, sucrose, hexose, and starch, followed by enhanced activities of sugar metabolism such as sucrose phosphate synthase, sucrose synthase, and invertase. Interestingly, expression of transcripts of enzymes involved in starch and sugar utilization were inhibited by EBR and H2O2. However, the effects of EBR on carbohydrate metabolisms were reversed by the H2O2 generation inhibitor diphenyleneodonium (DPI) or scavenger dimethylthiourea (DMTU) pretreatment. All of these results indicate that H2O2 functions as a secondary messenger for EBR-induced CO2 assimilation and carbohydrate metabolism in cucumber plants. Our study confirms that H2O2 mediates the regulation of photosynthesis by BRs and suggests that EBR and H2O2 regulate Calvin cycle and sugar metabolism via redox signaling and thus increase the photosynthetic potential and yield of crops.
PMCID: PMC3468824  PMID: 23024048
Metabolism; Photosynthesis; Reactive oxygen species; Rubisco; Sucrose
10.  Temperature- and concentration-dependence of kainate-induced γ oscillation in rat hippocampal slices under submerged condition 
Acta Pharmacologica Sinica  2012;33(2):214-220.
Fast neuronal network oscillation at the γ frequency band (γ oscillation: 30–80 Hz) has been studied extensively in hippocampal slices under interface recording condition. The aim of this study is to establish a method for recording γ oscillation in submerged hippocampal slices that allows simultaneously monitoring γ oscillation and the oscillation-related intracellular events, such as intracellular Ca2+ concentration or mitochondrial membrane potentials.
Horizontal hippocampal slices (thickness: 300 μm) of adult rats were prepared and placed in a submerged or an interface chamber. Extracellular field recordings were made in the CA3c pyramidal layer of the slices. Kainate, an AMPA/kainate receptor agonist, was applied via perfusion. Data analysis was performed off-line.
Addition of kainate (25–1000 nmol/L) induced γ oscillation in both the submerged and interface slices. Kainate increased the γ power in a concentration-dependent manner, but the duration of steady state oscillation was reduced at higher concentrations of kainate. Long-lasting γ oscillation was maintained at the concentrations of 100–300 nmol/L. Under submerged condition, γ oscillation was temperature-dependent, with the maximum power achieved at 29 °C. The induction of γ oscillation under submerged condition also required a fast rate of perfusion (5–7 mL/min) and showed a fast dynamic during development and after the washout.
The kainite-induced γ oscillation recorded in submerged rat hippocampal slices is useful for studying the intracellular events related to neuronal network activities and may represent a model to reveal the mechanisms underlying the normal neuronal synchronizations and diseased conditions.
PMCID: PMC4010343  PMID: 22266729
γ oscillation; neuronal network; hippocampus; kainite; submerged slice
11.  Effects of nitrogen form on growth, CO2 assimilation, chlorophyll fluorescence, and photosynthetic electron allocation in cucumber and rice plants*  
Cucumber and rice plants with varying ammonium (NH4 +) sensitivities were used to examine the effects of different nitrogen (N) sources on gas exchange, chlorophyll (Chl) fluorescence quenching, and photosynthetic electron allocation. Compared to nitrate (NO3 −)-grown plants, cucumber plants grown under NH4 +-nutrition showed decreased plant growth, net photosynthetic rate, stomatal conductance, intercellular carbon dioxide (CO2) level, transpiration rate, maximum photochemical efficiency of photosystem II, and O2-independent alternative electron flux, and increased O2-dependent alternative electron flux. However, the N source had little effect on gas exchange, Chl a fluorescence parameters, and photosynthetic electron allocation in rice plants, except that NH4 +-grown plants had a higher O2-independent alternative electron flux than NO3 −-grown plants. NO3 − reduction activity was rarely detected in leaves of NH4 +-grown cucumber plants, but was high in NH4 +-grown rice plants. These results demonstrate that significant amounts of photosynthetic electron transport were coupled to NO3 − assimilation, an effect more significant in NO3 −-grown plants than in NH4 +-grown plants. Meanwhile, NH4 +-tolerant plants exhibited a higher demand for the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for NO3 − reduction, regardless of the N form supplied, while NH4 +-sensitive plants had a high water-water cycle activity when NH4 + was supplied as the sole N source.
PMCID: PMC3030957  PMID: 21265044
Nitrogen form; Photosynthetic electron allocation; Alternative electron flux; Nitrate reductase
12.  Sequence features associated with microRNA strand selection in humans and flies 
BMC Genomics  2009;10:413.
During microRNA (miRNA) maturation in humans and flies, Drosha and Dicer cut the precursor transcript, thereby producing a short RNA duplex. One strand of this duplex becomes a functional component of the RNA-Induced Silencing Complex (RISC), while the other is eliminated. While thermodynamic asymmetry of the duplex ends appears to play a decisive role in the strand selection process, the details of the selection mechanism are not yet understood.
Here, we assess miRNA strand selection bias in humans and fruit flies by analyzing the sequence composition and relative expression levels of the two strands of the precursor duplex in these species. We find that the sequence elements associated with preferential miRNA strand selection and/or rejection differ between the two species. Further, we identify another feature that distinguishes human and fly miRNA processing machinery: the relative accuracy of the Drosha and Dicer enzymes.
Our result provides clues to the mechanistic aspects of miRNA strand selection in humans and other mammals. Further, it indicates that human and fly miRNA processing pathways are more distinct than currently recognized. Finally, the observed strand selection determinants are instrumental in the rational design of efficient miRNA-based expression regulators.
PMCID: PMC2751786  PMID: 19732433
13.  Poly[4-(dimethyl­amino)pyridinium [(μ6-5-carboxy­benzene-1,2,4-tricarboxy­ato-κ6 O 1:O 1′:O 2:O 4:O 4′:O 5)diargentate(I)]] 
In the title compound, {(C7H11N2)[Ag2(C10H3O8)]}n, the polymeric anion consists of two AgI atoms and a Hbtc3− ligand (H4btc = benzene-1,2,4,5-tetra­carboxylic acid). Each AgI atom is coordinated by four O atoms from three different Hbtc3− ligands. The two AgI atoms are bridged by two bidentate carboxyl­ate groups into an Ag2O4 cyclic unit, with an Ag⋯Ag distance of 2.8189 (3) Å. In this way, the Ag atoms are connected by the Hbtc3− ligands into an extended two-dimensional layer structure. A three-dimensional network is accomplished through O—H⋯O hydrogen bonds between the anionic layers. The cationic guest Hdmap+ [dmap = 4-(dimethyl­amino)pyridine] is trapped in the network and adheres to the layer by an N—H⋯O hydrogen bond.
PMCID: PMC2977362  PMID: 21583434
14.  A role of brassinosteroids in early fruit development in cucumber 
Journal of Experimental Botany  2008;59(9):2299-2308.
Brassinosteroids (BRs) are essential for many biological processes in plants, however, little is known about their roles in early fruit development. To address this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, cell division, and expression of cyclin and cyclin-dependent kinases (CDKs) genes were examined in two cucumber cultivars that differ in parthenocarpic capacity. The application of EBR induced parthenocarpic growth accompanied by active cell division in Jinchun No. 4, a cultivar without parthenocarpic capacity, whereas Brz treatment inhibited fruit set and, subsequently, fruit growth in Jinchun No. 2, a cultivar with natural parthenocarpic capacity, and this inhibitory effect could be rescued by the application of EBR. RT-PCR analysis showed both pollination and EBR induced expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB) after anthesis. cDNA sequences for CsCycD3;1 and CsCycD3;2 were isolated through PCR amplification. Both CsCycD3;1 and CsCycD3;2 transcripts were up-regulated by EBR treatment and pollination but strongly repressed by Brz treatment. Meanwhile, BR6ox1 and SMT transcripts, two genes involved in BR synthesis, exhibited feedback regulation. These results strongly suggest that BRs play an important role during early fruit development in cucumber.
PMCID: PMC2423651  PMID: 18515830
Brassinosteroids; cell division; Cucumis sativus; cyclin; flow cytometry; parthenocarpy

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