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The presence of pesticide residues in fresh fruits and vegetables poses a serious threat to human health. Brassinosteroids (BRs) can reduce pesticide residues in plants, but the underlying mechanisms still remain unclear. Here, we identified a tomato glutaredoxin gene GRXS25 which was induced by 24-epibrassinolide (EBR) and chlorothalonil (CHT) in a way dependent on apoplastic reactive oxygen species (ROS). Silencing of GRXS25 in tomato abolished EBR-induced glutathione S-transferases (GSTs) gene expression and activity, leading to an increased CHT residue. Yeast two-hybrid and bimolecular fluorescence complementation assays showed protein-protein interaction between GRXS25 and a transcription factor TGA2. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that TGA2 factor bound to the TGACG-motif in the GST3 promoter. While silencing of TGA2 strongly compromised, overexpression of TGA2 enhanced expression of GST genes and CHT residue metabolism. Our results suggest that BR-induced apoplastic ROS trigger metabolism of pesticide residue in tomato plants through activating TGA2 factor via GRXS25-dependent posttranslational redox modification. Activation of plant detoxification through physiological approaches has potential implication in improving the food safety of agricultural products.

Melatonin is a pleiotropic signaling molecule that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms for melatonin-mediated thermotolerance remain largely unknown. Here, we report that endogenous melatonin levels increased with a rise in ambient temperature and that peaked at 40°C. Foliar pretreatment with an optimal dose of melatonin (10 μmol/L) or the overexpression of N-acetylserotonin methyltransferase (ASMT) gene effectively ameliorated heat-induced photoinhibition and electrolyte leakage in tomato plants. Both exogenous melatonin treatment and endogenous melatonin manipulation by overexpression of ASMT decreased the levels of insoluble and ubiquitinated proteins, but enhanced the expression of heat-shock proteins (HSPs) to refold denatured and unfolded proteins under heat stress. Meanwhile, melatonin also induced expression of several ATG genes and formation of autophagosomes to degrade aggregated proteins under the same stress. Proteomic profile analyses revealed that protein aggregates for a large number of biological processes accumulated in wild-type plants. However, exogenous melatonin treatment or overexpression of ASMT reduced the accumulation of aggregated proteins. Aggregation responsive proteins such as HSP70 and Rubisco activase were preferentially accumulated and ubiquitinated in wild-type plants under heat stress, while melatonin mitigated heat stress-induced accumulation and ubiquitination of aggregated proteins. These results suggest that melatonin promotes cellular protein protection through induction of HSPs and autophagy to refold or degrade denatured proteins under heat stress in tomato plants.

The high-resolution crystal structure of a complex of the lactonohydrolase zearalenone hydrolase with the hydrolytic product of the oestrogenic myotoxin zearalenone provides information on the final stage of the catalytic mechanism., Zearalenone hydrolase (ZHD) is an α/β-hydrolase that detoxifies and degrades the lactone zearalenone (ZEN), a naturally occurring oestrogenic mycotoxin that contaminates crops. Several apoenzyme and enzyme–substrate complex structures have been reported in the resolution range 2.4–2.6 Å. However, the properties and mechanism of this enzyme are not yet fully understood. Here, a 1.60 Å resolution structure of a ZHD–product complex is reported which was determined from a C-terminally His6-tagged ZHD crystal soaked with 2 mM ZEN for 30 min. It shows that after the lactone-bond cleavage, the phenol-ring region moves closer to residues Leu132, Tyr187 and Pro188, while the lactone-ring region barely moves. Comparisons of the ZHD–substrate and ZHD–product structures show that the hydrophilic interactions change, especially Trp183 N∊1, which shifts from contacting O2 to O12′, suggesting that Trp183 is responsible for the unidirectional translational movement of the phenol ring. This structure provides information on the final stage of the catalytic mechanism of zearalenone hydrolysis.

Use of antibiotic-contaminated manure in crop production poses a severe threat to soil and plant health. However, few studies have studied the mechanism by which plant development is affected by antibiotics. Here, we used microscopy, flow cytometry, gene expression analysis and fluorescent dyes to study the effects of oxytetracycline (OTC), a widely used antibiotic in agriculture, on root meristem activity and the accumulation of hydrogen peroxide (H2O2) and nitric oxide (NO) in the root tips of tomato seedlings. We found that OTC caused cell cycle arrest, decreased the size of root meristem and inhibited root growth. Interestingly, the inhibition of root growth by OTC was associated with a decline in H2O2 levels but an increase in NO levels in the root tips. Diphenyliodonium (DPI), an inhibitor of H2O2 production, showed similar effects on root growth as those of OTC. However, exogenous H2O2 partially reversed the effects on the cell cycle, meristem size and root growth. Importantly, cPTIO (the NO scavenger) and tungstate (an inhibitor of nitrate reductase) significantly increased H2O2 levels in the root tips and reversed the inhibition of root growth by OTC. Out results suggest that OTC-induced NO production inhibits H2O2 accumulation in the root tips, thus leading to cell cycle arrest and suppression of root growth.

Ethylene responsive factors (ERFs) act as critical downstream components of the ethylene signalling pathway in regulating plant development and stress responses. However little is known about its role in regulation of photosynthesis. Here, we identified an ethylene-inducible ERF gene in citrus, CitERF13. Transient over-expression of CitERF13 in N. tabacum leaves, resulted in a significant decrease in net photosynthetic rate. Closer examination of photosynthetic activity of PSII and PSI indicated that CitERF13 overexpression led to declines of Fv/Fm, Y(II) and Y(I). However, change in NPQ was less pronounced. CitERF13 overexpression also significantly reduced Vc,max, Jmax and AQY, indicating inhibition of the Calvin cycle. The expression of photosynthesis-related genes was suppressed to a variable extent in leaf blades transiently over-expressing CitERF13. CitERF13 transient overexpression in tobacco or citrus both resulted in a decline of Chlorophyll content and CitERF13 overexpressing tobacco leaf disc was more susceptible to chlorosis in response to MV-mediated oxidative stress. The results suggest that CitERF13 is potentially involved in suppressing photosynthesis through multiple pathways, for instance, inhibiting photochemical activity of photosynthesis, CO2 carboxylation capacity and chlorophyll metabolism.

The study investigated the effects and underlying mechanisms of silent information regulation of transcription 1 (Sirt1) action on apoptosis in chondrocytes and degradation of the extracellular matrix. Cartilage tissue samples were derived from knee arthroplasty of patients with osteoarthritis (OA). The three groups were as follows: Control, resveratrol (Res) and Res+small interfering (si)RNA (Res+siRNA Sirt1). The level of Sirt1 protein expression significantly increased in the Res group (1.03±0.10) compared with the control (0.22±0.03) and Res+siRNA (0.18±0.01) groups (both P

• Brassinosteroids (BRs) are a new class of plant hormones that are essential for plant growth and development. Here, the involvement of BRs in plant systemic tolerance to biotic and abiotic stresses was studied. • The effects of 24-epibrassinolide (EBR) on plant stress tolerance were studied through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation, the analysis of gene expression using quantitative real-time PCR and the measurement of hydrogen peroxide (H₂O₂) production using a spectrophotometric assay or confocal laser scanning microscopy. • Treatment of primary leaves with EBR induced systemic tolerance to photooxidative stress in untreated upper and lower leaves. This was accompanied by the systemic accumulation of H₂O₂ and the systemic induction of genes associated with stress responses. Foliar treatment of EBR also enhanced root resistance to Fusarium wilt pathogen. Pharmacological study showed that EBR-induced systemic tolerance was dependent on local and systemic H₂O₂ accumulation. The expression of BR biosynthetic genes was repressed in EBR-treated leaves, but elevated significantly in untreated systemic leaves. Further analysis indicated that EBR-induced systemic induction of BR biosynthetic genes was mediated by systemically elevated H₂O₂. • These results strongly argue that local EBR treatment can activate the continuous production of H₂O₂, and the autopropagative nature of the reactive oxygen species signal, in turn, mediates EBR-induced systemic tolerance.

To determine whether light quality affects the incidence of disease, we exposed cucumber (Cucumis sativus L. cv. Jinyan No. 4) plants at the 4-leaf stage to white and other monochromatic lights and tested the effects on plant response to Sphaerotheca fuliginea, defence-related gene expression and metabolic changes. Exposure to red light resulted in higher levels of H2O2 and salicylic acid (SA), and stronger expression of defence genes such as PR-1 than exposure to white or other monochromatic lights. In comparison, plants grown under purple and blue light had higher activities of phenylalanine ammonia-lyase (PAL) and polyphenoloxidase (PPO) and higher level of flavonoids than plants grown under other lights. Furthermore, plants grown under red light were more resistant whilst plants grown under other monochromatic lights were less resistant to Sphaerotheca fuliginea than plants grown under white light. These results suggest a role of red light in light-enhanced resistance, which correlates with enhanced SA-dependent signaling pathway.

Previously, in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and another kind of cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and Annexin A2 shown as one of the significantly up-regulated proteins accompanying cellular transformation. Annexin A2 was further validated for its expression in the three kinds of cells and in the clinical samples of tumour tissues and their adjacent normal epithelia from primary OSCC patients. Western blot analysis and real-time PCR detected increased Annexin A2 protein and mRNA levels in cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry showed elevated Annexin A2 protein expression in tumour tissues over the adjacent non-malignant epithelia from OSCC patients; however, the mRNA levels between tumour and normal tissues did not change significantly. Interestingly, levels of Annexin A2 protein expression negatively correlated with the tumour differentiation grades. The results presented here suggest that Annexin A2 protein may play important roles in carcinogenesis of OSCC, and it may also serve as a candidate biomarker for pathologic differentiation grade of OSCC.

Background The present study was designed to examine and analyze the global gene expression changes during the tumorigenesis of a human immortalized oral epithelial cell line, and search for the possible genes that may play a role in the carcinogenesis of oral cancer associated with benzo (a) pyrene. Methods The human immortalized oral epithelial cells, which have been established through transfection of E6/E7 genes of human papillomavirus type 16 and proved to be non-tumorigenic in nude mice, were treated with benzo (a) pyrene. Tumorigenesity of the treated cells were examined through nude mice subcutaneous injection. The global gene expression profiles of immortalized cells and the tumorigenic cells were acquired through hybridization of a microarray of Affymetrix U133 plus 2.0. The data were analyzed using Spring 7.0 software and treated statistically using one-way analysis of variance (ANOVA). The differentially expressed genes were classified using a Venn diagram and annotated with gene ontology. Several highlighted genes were validated in cells using a real-time polymerase chain reaction. Results There were 883 differentially expressed genes during the tumorigenesis and most of them changed expression in the early stage of tumorigenesis. These genes mainly involved in macromolecule metabolism and signal transduction, possessed the molecular function of transition metal ion binding, nucleotide binding and kinase activity; their protein products were mainly integral to membranes or localized in the nucleus and cytoskeleton. The expression patterns of IGFBP3, S100A8, MAP2K, KRT6B, GDF15, MET were validated in cells using a real-time polymerase chain reaction; the expression of IGFBP3 was further validated in clinical oral cancer specimens. Conclusions This study provides the global transcription profiling associated with the tumorigenesis of oral epithelial cells exposed to benzo (a) pyrene; IGFBP3 may play a potential role in the initiation of oral cancer related to benzo (a) pyrene exposure.

Percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] has been developed into a mature interventional treatment for atherosclerotic cardiovascular disease. However, the long-term therapeutic effect is compromised by the high incidence of vascular restenosis following angioplasty, and the underlying mechanisms of vascular restenosis have not yet been fully elucidated. In the present study, we investigated the role of the gap junction (GJ) protein, connexin 43 (Cx43), in the development of vascular restenosis. To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty injury. At 0, 7, 14 and 2 days following balloon injury, the arteries were removed, and the intimal/medial area of the vessels was measured to evaluate the degree of restenosis. We found that the intimal area gradually increased following balloon injury. Intimal hyperplasia and restenosis were particularly evident at 14 and 28 days after injury. In addition, the mRNA and protein expression of Cx43 was temporarily decreased at 7 days, and subsequently increased at 14 and 28 days following balloon injury, as shown by RT-PCR and western blot analysis. To determine the involvement of Cx43 in vascular restenosis, the lentivirus vector expressing shRNA targeting Cx43, Cx43-RNAi-LV, was used to silence Cx43 in the rat carotid arteries. The knockdown of Cx43 effectively attenuated the development of intimal hyperplasia and vascular restenosis following balloon injury. Thus, our data indicate the vital role of the GJ protein, Cx43, in the development of vascular restenosis, and provide new insight into the pathogenesis of vascular reste-nosis. Cx43 may prove to be a novel potential pharmacological target for the prevention of vascular restenosis following PCI.

Salicylic acid (SA) plays a critical role in plant defense against pathogen attack. The SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense, which is pathogenesis-related protein-independent but involves an RNA-dependent RNA polymerase 1 (RDR1)-mediated RNA silencing mechanism and/or an alternative oxidase (AOX)-associated defense pathway. However, the relationship between these two viral defense-related pathways remains unclear. In this study, Tobacco mosaic virus (TMV) inoculation onto Solanum lycopersicum (tomato) leaves induced a rapid induction of the SlAOX1a transcript level as well as the total and CN-resistant respiration at 0.5 dpi, followed by an increase in SlRDR1 gene expression at 1 dpi in the upper uninoculated leaves. Silencing SlRDR1 using virus-induced gene silencing system significantly reduced SlRDR1 expression and tomato defense against TMV but had no evident effect on SlAOX1a transcription. Conversely, silencing SlAOX1a not only effectively reduced the AOX1a transcript level, but also blocked the TMV-induced SlRDR1 expression and decreased the basal defense against TMV. Furthermore, the application of an exogenous AOX activator on empty vector-silenced control plants greatly induced the accumulation of SlRDR1 and SlAOX1a transcript and reduced TMV viral RNA accumulation, but failed to have such effects on SlRDR1-silenced plants. Moreover, RDR1-overexpressed transgenic Nicotiana benthamiana plants enhanced defense against TMV than the empty vector-transformed plants, but these effects were not affected by the exogenous AOX activator or inhibitor. These results indicate that RDR1 is involved in the AOX-mediated defense pathway against TMV infection and plays a crucial role in enhancing RNA silencing to limit virus systemic spread.

Spinal cord injury (SCI)‑induced osteoporosis may cause mild trauma to bone and increase the risk of bone fracture. The present study aimed to investigate the efficacy of coenzyme Q (CoQ10) on SCI‑induced osteoporosis in rats. SCI was induced by surgical transection of the cord at the T10‑12 level. Animals were treated with CoQ10 (10 mg/kg; intragastrically) daily from 12 h after the surgery and over 10 subsequent days. At the end of the experimental period, blood was collected from the animals and femurs and tibiae were removed for evaluation using biochemical assays. Treatment with CoQ10 prevented SCI‑induced bone loss by rescuing the decreased levels of bone mineral density and bone mineral content observed in the SCI rats. Furthermore, CoQ10 administration reduced bone malondialdehyde levels with a concomitant increase in superoxide dismutase levels, thus alleviating SCI‑induced oxidative injury. In addition, serum inflammatory cytokine levels were markedly increased in rats post‑SCI, which was attenuated by treatment with CoQ10. Finally, the osteoclast‑specific genes receptor activator of nuclear factor kappa‑B ligand and cathepsin K were significantly upregulated and the osteoblast‑specific gene core‑binding factor alpha 1 in the femur was downregulated following SCI, which was effectively restored following treatment with CoQ10. The results suggested that CoQ10 treatment may be effective in attenuating SCI‑induced osteoporosis.

Activity of voltage-gated K+ channels (KV) in pulmonary arterial smooth muscle cells (PASMC) is pivotal in controlling membrane potential, cytoplasmic free Ca2+ concentration ([Ca2+]cyt, and pulmonary vasomotor tone. Acute hypoxia selectively inhibits KV channels, depolarizes PASMC, raises [Ca2+]cyt, and causes pulmonary vasoconstriction and vascular remodeling. Prolonged hypoxia (24-60 h) decreased significantly the mRNA levels of KV channel alpha subunits, KV1.2 and KV1.5. Consistently, the protein levels of KV1.2 and KV1.5 were also decreased significantly by hypoxia (48-72 h). Nevertheless, hypoxia affected negligibly the mRNA levels of KV channel beta subunits (KVbeta1, KVbeta2, and KVbeta3). The native K+ channels are composed of pore-forming alpha and auxiliary beta subunits. Assembly of KV beta subunits with alpha subunits confers rapid inactivation on the slowly or non-inactivating delayed rectifier KV channels. KV beta subunits also function as an open-channel blocker of KV channels. Thus, the diminished transcription and expression of KV alpha subunits may reduce the number of KV channels and decrease KC currents. Unchanged transcription of KV beta subunits may increase the fraction of the KV channel alpha subunits that are associated with beta subunits and further reduce the total KV currents. These data demonstrate a novel mechanism by which chronic hypoxia may cause pulmonary vasoconstriction and hypertension.

A combination of molecular-targeted cancer imaging and therapy is an emerging strategy to improve cancer diagnosis and minimize the side effects of conventional treatments. Here, we generated a recombinant protein, EC1-GLuc-p53C, by fusing EC1 peptide, an artificial ligand of ErbB2, with Gaussia luciferase (GLuc) and a p53-activating peptide, p53C. EC1-GLuc-p53C was expressed and purified from E. coli BL21. In vitro experiments showed that EC1-GLuc-p53c was stable in luminescent activity and selectively targeted ErbB2-overexpressing BT474 cells for bioluminescence imaging. Moreover, the internalized EC1-GLuc-p53C in BT474 cells exerted its function to reactivate p53 and significantly inhibited cellular proliferation. In tumor-bearing mice, the ErbB2-targeted bioluminescence imaging and therapeutic effect of EC1-GLuc-p53C were also observed specifically in BT474 tumors but not in MCF7 tumors, which does not overexpress ErbB2. Thus, the present study demonstrates EC1-GLuc-p53C to be an effective theranostic reagent targeting ErbB2 for bioluminescence imaging and cancer therapy.

High-throughput tag-sequencing (Tag-seq) analysis based on the Solexa Genome Analyzer platform was applied to analyze the gene expression profiling of cucumber plant at 5 time points over a 24h period of waterlogging treatment. Approximately 5.8million total clean sequence tags per library were obtained with 143013 distinct clean tag sequences. Approximately 23.69%–29.61% of the distinct clean tags were mapped unambiguously to the unigene database, and 53.78%–60.66% of the distinct clean tags were mapped to the cucumber genome database. Analysis of the differentially expressed genes revealed that most of the genes were down-regulated in the waterlogging stages, and the differentially expressed genes mainly linked to carbon metabolism, photosynthesis, reactive oxygen species generation/scavenging, and hormone synthesis/signaling. Finally, quantitative real-time polymerase chain reaction using nine genes independently verified the tag-mapped results. This present study reveals the comprehensive mechanisms of waterlogging-responsive transcription in cucumber.

Plant phosphate transporter (PT) genes comprise a large family with important roles in various physiological and biochemical processes. In this study, a database search yielded 26 potential PT family genes in rice (Oryza sativa). Analysis of these genes led to identification of eight conserved motifs and 5-12 trans-membrane segments, most of them conserved. A total of 237 putative cis elements were found in the 2-kb upstream region of these genes. Of these, a majority were Pi-response and other stress-related cis regulatory elements, such as PHO-like, TATA-box-like, PHR1, or Helix-loop-helix elements, and WRKY1 and ABRE elements, suggesting gene regulation by these signals. Comprehensive expression analysis of these genes was performed using data from microarrays hybridized with RNA from 27 tissues covering the entire lifecycle from three rice genotypes: Minghui 63, Zhenshan 97, and Shanyou 63. Real-time PCR analysis confirmed that three rice PT genes are preferentially expressed in stamen at 1 d before flowering, two in panicle at the heading stage, and two in flag leaf at 14 d after the heading stage. Hormone-treatment experiments revealed differential up-regulation or down-regulation of 11 rice PT genes in seedlings exposed to five hormones, respectively. These results will be useful for elucidating the roles of these genes in the growth, development, and stress response of the rice plant.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has >50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5ʹ-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36–37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000–180,000 Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues. Compared with rat protein, human IRS-1 contains more potential Ser/Thr phosphorylation sites and only one nucleotide binding site. The entire protein coding sequence is contained within a single exon.

Brassinosteroids (BRs) are known to protect crops from the toxicity of herbicides, fungicides and insecticides. It is shown here that application of 24-epibrassinolide (EBR) accelerated metabolism of various pesticides and consequently reduced their residual levels in cucumber ( Cucumis sativus L). Chlorpyrifos, a widely used insecticide, caused significant reductions of net photosynthetic rate (Pn) and quantum yield of PSII (Phi(PSII)) in cucumber leaves. EBR pretreatment alleviated the declines of Pn and Phi(PSII) caused by chlorpyrifos application, and this effect of EBR was associated with reductions of chlorpyrifos residues. To understand how EBR promotes chlorpyrifos metabolism, the effects of EBR on activity and expression of enzymes involved in pesticide metabolism were analyzed. EBR had a positive effect on the activation of glutathione S-transferase (GST), peroxidase (POD), and glutathione reductase (GR) after treatment with chlorpyrifos, although the effect on GR was attenuated at later time points when plants were treated with 1 mM chlorpyrifos. In addition, EBR enhanced the expression of P450 and MRP, which encode P450 monooxygenase and ABC-type transporter, respectively. However, the expression of GST was consistently lower than that of plants treated with only chlorpyrifos. Importantly, the stimulatory effect of EBR on pesticide metabolism was also observed for cypermethrin, chlorothalonil, and carbendazim, which was attributed to the enhanced activity and genes involved in pesticide metabolism. The results suggest that BRs may be promising, environmentally friendly, natural substances suitable for wide application to reduce the risks of human and environment exposure to pesticides.

Several benzoic and cinnamic acid derivatives were identified from cucumber root exudates. The effects of these phenylcarboxylic acids on root growth and cell cycle progression were examined in germinated seeds of cucumber. All 12 phenylcarboxylic acids (0.25 mM) tested significantly inhibited cucumber radicle growth, and cinnamic acid exerted a dose-dependent inhibitory effect. At 6 h after exposure to the acids, transcript levels of the cell cycle-related genes, including two cyclin-dependent kinases (CDKs) and four cyclins were reduced. Among them, transcript of CycB, a marker gene for mitosis showed a remarkable reduction. The temporal analysis showed that expression of mitotic genes (CDKB, CycA, and CycB) were reduced throughout the experiment, while the reduction of the other genes (CDKA, CycD3;1, and CycD3;2) were observed only at earlier time points. At 48 h after treatment with benzoic and cinnamic acids, an enhancement of endoreduplication was observed. Further time course analysis indicated that endoreduplication started as early as 6 h after exposure to cinnamic acid. These results provide evidence that exposure to benzoic and cinnamic acids can induce rapid and dramatic down-regulation of cell cycle-related genes, thus leading to root growth inhibition. Meanwhile, the block of mitosis caused by phenylcarboxylic acids also induced an increased level of endoreduplication.

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2-DE and LC-tandem mass chromatography to separate and identify differentially expressed proteins. Forty-five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.

We investigated in vivo the effect of recombinant bactericidal/permeability-increasing protein (rBPI21) on high-mobility group box 1 protein (HMGB1) expression in sepsis and its potential mechanism. Using a sepsis model induced by cecal ligation and puncture (CLP), rats were randomly divided into four groups as follows: normal control group, sham-operated group, CLP group, and BPI treatment group. Animals were killed at designated time points, and blood and tissue samples from liver, lungs, kidneys, and small intestine were harvested to determine related variables. In addition, we observed the effect of treatment with rBPI21 on survival rate in septic rats. The results showed that endotoxin content and expression levels of HMGB1 and LPS binding protein/CD14 mRNA in various organs were significantly increased at 12 and 24 h after CLP, which can be attenuated by treatment with rBPI21 (P

Substrate topography is one of the key factors that influence cell behavior, such as cell attachment, adhesion, proliferation and differentiation. In the present work, nanostructures were produced on polystyrene Petri dish by polarized laser irradiation with the wavelength of 266 nm and the energy of 3.0 mJ/cm2. Cell adhesion, growth and gene expression of Madine darby canine kidney (MDCK) cells cultured on smooth and nanogrooved substrates were investigated. The results indicated that cells preferred to adhere and grow on nanogrooved substrate. The distribution of cell cycle for cells on smooth substrates was different from that on nanogrooved substrate. The percentage of G1 phase cells on nanogrooved substrate (48.6 +/- 1.4%) was lower than that on smooth substrate (57.6 +/- 4.4%), while the percentage of cells on nanogrooved substrate in S (30.2 +/- 0.5%) and G2/M (21.2 +/- 1.1%) phase was higher than those on smooth substrate (25.1 +/- 1.5% and 17.3 +/- 3.3%, respectively). Moreover, the gene expression of cyclin D1 and keratin 18, which was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), was significantly enhanced by nanogrooves, with an increase of cyclin D1 mRNA by 98% and an increase of keratin 18 mRNA by 75%. In conclusion, the nanogrooved surface features on polystyrene could alter cell cycle and enhance gene expression of cyclin D1 and keratin 18 in MDCK cells, which partly explained the increased cell adhesion and growth on nanogrooved substrate.

Alopecia is a predominant feature of vitamin D receptor inactivation in mice and humans. To determine the role of vitamin D receptor in the regulation of hair growth directly, we used the human keratin 14 promoter to target human vitamin D receptor expression to the skin of transgenic mice, and generated vitamin D receptor null mice that express the human vitamin D receptor transgene. Parallel studies were carried out in littermates of wild-type, vitamin D receptor null, transgenic, and human vitamin D receptor-expressing null mice in two transgenic lines. The transgenic mice were grossly normal. The vitamin D receptor null and vitamin D receptor null/human vitamin D receptor mice were growth retarded and developed hypocalcemia, secondary hyperparathyroidism, and rickets. In contrast to the vitamin D receptor null mice that developed alopecia, however, the vitamin D receptor null/human vitamin D receptor mice displayed a normal hair coat, and their hair shaft and skin histology were indistinguishable from those of the wild-type mice. Immunohistochemical analyses revealed that the human vitamin D receptor was highly expressed in the basal layer of the epidermis and outer root sheath of the hair follicle. During follicular morphogenesis, no major histologic differences were seen in the skin of wild-type, vitamin D receptor null, transgenic, and vitamin D receptor null/human vitamin D receptor littermates. When anagen was induced by hair depilation at day 20 after birth, the vitamin D receptor null mice failed to initiate the hair cycle, whereas the vitamin D receptor null/human vitamin D receptor mice displayed the same pattern of anagen follicle formation as the wild-type mice. Interestingly, the transgenic mice initiated the follicular cycle earlier than the wild-type and vitamin D receptor null/human vitamin D receptor mice in a gene concentration-dependent manner. Taken together, these data provide direct evidence that vitamin D receptor is required for the initiation of the postnatal hair follicular cycle in mice.

Transcription factors are frequently altered in leukemia through chromosomal translocation, mutation or aberrant expression. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukemia (AML), is a transcription factor implicated in both gene repression and activation. AML1-ETO exists as dimer/oligomer through its NHR2 dimerization domain. Given that the AML1-ETO dimerization has been shown to be critical for its leukemogenic activity, it is important to identify coregulatory factors that “read” the NHR2 dimerization and contribute to leukemogenesis. We now show that, in leukemic cells, AML1-ETO resides in and functions through a stable protein complex (AETFC) that contains several hematopoietic transcription factors and cofactors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, colocalize genome-wide, cooperatively regulate gene expression, and contribute to leukemogenesis. Within the AETFC complex, the AML1-ETO dimerization is required for a specific interaction between the dimerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic study of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric transcription factors create a new protein-binding interface through dimerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced hematopoietic stem cell self-renewal and leukemogenesis. These results reveal new mechanisms of action of AML1-ETO in leukemogenesis and provide potential therapeutic possibilities for targeting t(8;21)+ AML. Citation Format: Xiao-Jian Sun, Zhanxin Wang, Lan Wang, Yanwen Jiang, T. David Soong, Nils Kost, Wei-Yi Chen, Olivier Elemento, Wolfgang Fischle, Ari Melnick, Dinshaw J. Patel, Stephen D. Nimer, Robert G. Roeder. A stable transcription factor complex nucleated by dimeric AML1-ETO controls leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-111. doi:10.1158/1538-7445.AM2013-LB-111