Significant Reduction In Protein Expression Research Articles

Antimicrobial impacts of zinc oxide nanoparticles on Shiga toxin-producing Escherichia coli (serotype O26)

Abstract The antibacterial activity of zinc oxide nanoparticles (ZnO NPs) has received significant attention worldwide due to the emergence of multidrug-resistant microorganisms. Shiga toxin-producing Escherichia coli is a major foodborne pathogen that causes gastroenteritis that may be complicated by hemorrhagic colitis or hemolytic uremic syndrome. Therefore, this study aimed to evaluate the antimicrobial effect of ZnO NPs against E. coli O26 and its Shiga toxin type 2 (Stx2). Multidrug resistance phenotype was observed in E. coli O26, with co-resistance to several unrelated families of antimicrobial agents. Different concentrations of ZnO NPs nanoparticles (20 nm) were tested against different cell densities of E. coli O26 (108, 106 and 105 CFU/ml). The minimum inhibitory concentration (MIC) value was 1 mg/ml. Minimum bactericidal concentration (MBC) was 1.5 mg/ml, 2.5 mg/ml and 3 mg/ml, respectively, depending on ZnO NPs concentrations and bacterial cell density. Results showed a significant (P≤0.05) decrease in Stx2 level in a response to ZnO NPs treatment. As detected by quantitative real-time PCR, ZnO NPs down-regulated the expression of the Stx2 gene (P≤0.05). Moreover, various concentrations of ZnO NPs considerably reduced the total protein content in E. coli O26. There was a significant reduction in protein expression with increased ZnO NPs concentration compared to the non-treated control. Scanning electron micrographs (SEM) of the treated bacteria showed severe disruptive effects on E. coli O26 with increasing ZnO NPs concentration. The results revealed a strong correlation between the antibacterial effect and ZnO NPs concentrations. ZnO NPs exert their antibacterial activities through various mechanisms and could be used as a potent antibacterial agent against E. coli O26.

Betaine Reduces Lipid Anabolism and Promotes Lipid Transport in Mice Fed a High-Fat Diet by Influencing Intestinal Protein Expression.

Betaine is more efficient than choline and methionine methyl donors, as it can increase nitrogen storage, promote fat mobilisation and fatty acid oxidation and change body fat content and distribution. Lipid is absorbed primarily in the small intestine after consumption, which is also the basis of lipid metabolism. This study was conducted to establish a mouse model of obesity in Kunming mice of the same age and similar body weight, and to assess the effect of betaine on the intestinal protein expression profile of mice using a proteomic approach. Analysis showed that betaine supplementation reversed the reduction in expression of proteins related to lipid metabolism and transport in the intestine of mice induced by a high-fat diet (HFD). For example, the addition of betaine resulted in a significant upregulation of microsomal triglyceride transfer protein (Mttp), apolipoprotein A-IV (Apoa4), fatty-acid-binding protein 1 (Fabp1) and fatty-acid-binding protein 2 (Fabp2) expression compared to the HFD group (p < 0.05), which exhibited accelerated lipid absorption and then translocation from the intestine into the body’s circulation, in addition to a significant increase in Acetyl-CoA acyltransferase (Acaa1a) protein expression, hastening lipid metabolism in the intestine (p < 0.05). Simultaneously, a significant reduction in protein expression of alpha-enolase 1 (Eno1) as the key enzyme for gluconeogenesis in mice in the betaine-supplemented group resulted in a reduction in lipid synthesis in the intestine (p < 0.05). These findings provide useful information for understanding the changes in the protein profile of the small intestine in response to betaine supplementation and the potential physiological regulation of diets’ nutrient absorption.

Thalidomide Inhibits Angiogenesis via Downregulation of VEGF and Angiopoietin-2 in Crohn's Disease.

Immune-mediated angiogenesis is important in the pathogenesis of inflammatory bowel disease and targeted treatment could alleviate the disease. Thalidomide is an effective drug in inflammatory bowel disease, which might be related to its multiple role in anti-inflammatory, immunoregulatory, and anti-angiogenesis. This study is to investigate the effect of thalidomide on angiogenesis in tissues from patients and in vitro cells. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), VEGF, and CD31 expressions in intestinal mucosa from pediatric CD patients before and after thalidomide treatment were measured by immunohistochemistry. Western blotting and polymerase chain reaction were performed to characterize the change of angiogenic factors before and after treatment in remission. Human umbilical vein endothelial cells (HUVECs) treated by thalidomide were used to examine its effect on endothelial cell proliferation and migration and capillary-like structures. Results showed that VEGF and Ang-2 levels were significantly greater in CD patients over controls. Thalidomide produced a significant reduction in protein expression of Ang-2 and VEGF, along with a decrease in mRNA expression of Ang-2. While, Ang-1 level did not show a statistically significant change. Thalidomide significantly inhibited cell proliferation in a dose-dependent manner. It also suppressed VEGF- and Ang-2-induced cell migration and capillary-like tube formation in HUVECs. Therefore, our study suggests that VEGF and Ang-2 levels are up-regulated in pediatric CD patients. It also indicated that thalidomide can be able to deactivate endothelium by the downregulation effect on angiogenic factors by targeting VEGF and Ang-2.

EXTH-21. DIFFERENTIAL PROTEIN EXPRESSION LEVELS OF ABC AND SOLUTE CARRIER TRANSPORTERS AT THE BLOOD-BRAIN BARRIER OF HUMAN BRAIN AND GLIOBLASTOMA

Abstract BACKGROUND Mechanistic understanding and quantitative prediction of drug penetration across the human blood-brain barrier (BBB) is critical to rational drug development and treatment for brain cancer especially glioblastoma. However, prediction of drug brain/tumor penetration has been significantly hindered mainly due to the lack of quantitation data on transporter protein expression levels at the human BBB. This study was to determine protein expression levels of major transporters and markers at the BBB of human brain and glioblastoma. METHOD The absolute protein expression levels of major transporters and markers were determined in isolated microvessels of human brain (N=30), glioblastoma (N=47), rat (N=10) and mouse brain (N=10), using liquid chromatography with tandem mass spectrometry (LC-MS/MS) based targeted proteomics. RESULTS In isolated microvessels of 30 human brain specimens, the median protein abundances for ABCB1, ABCG2, GLUT1, GLUT3, LAT1, MCT1, Na/K ATPase, and Claudin-5 were 3.38, 6.21, 54.51, 7.17, 3.42, 5.71, 32.14, and 1.15 fmol/µg protein, respectively. In glioblastoma microvessels, ABCB1, ABCG2, MCT1, GLUT1, Na/K ATPase, and Claudin-5 protein levels were significantly reduced, while LAT1 was increased and GLU1 remained the same. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in isolated microvessels of both human brain and glioblastoma. There was species difference in transporter protein expression levels in isolated microvessels of human, rat and mouse brain. Specifically, rodent BBB expressed significantly higher ABCB1 but similar ABCG2, as compared to human BBB. CONCLUSION The physical and biochemical barriers of the BBB in glioblastomas are largely disrupted, as indicated by the loss or significant reduction in protein expression of the tight junction marker (claudin-5), brain endothelial cell marker (GLUT1), and major efflux transporters (ABCB1 and ABCG2) as compared to normal human BBB. Differential BBB transporter protein expression levels provides mechanistic and quantitative basis for the prediction of heterogeneous drug penetration into human normal brain and glioblastoma.

Nanoparticulate TiO₂ Induced Suppression of Spermatogenesis is Involved in Regulatory Dysfunction of the cAMP-CREB/CREM Signaling Pathway in Mice.

Long-term exposure to nanoparticulate titanium dioxide (nano-TiO₂) is known to cause reductions of sperm numbers and quality in animals, and the cAMP-dependent signaling pathway has been demonstrated to play a key role in regulating spermatogenesis. However, whether the suppression of spermatogenesis induced by nano-TiO₂ is related to regulatory disturbances of the cAMP-CREB/CREM signaling pathway is not well investigated. In the current study, male mice were exposed to nano-TiO₂ at doses of 1.25, 2.5, or 5 mg/kgbw via gavage instillation for 90 consecutive days and the molecular mechanisms underlying suppression of spermatogenesis caused by nano-TiO₂ were investigated. Our findings showed that nano-TiO₂ could cross the blood-testis barrier, and accumulated in mouse testes, thus inducing obvious pathological changes and decreasing sperm concentrations and motility, as well as increasing rate of sperm malformation. Furthermore, nano-TiO₂ also induced significant reductions in protein expression including cyclic adenosine monophosphate content, protein kinase A, cAMP-responsive element modulator, p-cAMP-response element binding protein, lactate dehydrogenase-C, testis-specific protein kinase 1, and testicular specific CREM activator, and upregulation of protein expression including protein phosphatase, and transducer of regulated CREB 1, which may be associated with reductions of follicle stimulating hormone and luteinizing hormone levels. Together, the present study indicates that the reductions of FSH and LH concentrations and suppression of spermatogenesis in mice caused by nano-TiO₂ may be associated with the dysfunctions of the cAMP-CREB/CREM signaling pathway.

Gold nanoparticle–conjugated quercetin inhibits epithelial–mesenchymal transition, angiogenesis and invasiveness via EGFR/VEGFR‐2‐mediated pathway in breast cancer

Epidermal growth factor plays a critical role in breast malignancies by enhancing cell proliferation, invasion, angiogenesis and metastasis. Epithelial-mesenchymal transition (EMT) is a crucial process by which epithelial cells lose polarity and acquire migratory mesenchymal properties. Gold nanoparticles are an efficient drug delivery vehicle for carrying chemotherapeutic agents to target cancer cells and quercetin is an anti-oxidative flavonoid known with potent anti-malignant cell activity. Cell viability was assessed by MTT assay, and protein expression was examined by Western blotting and immunocytochemistry. Cell invasion was monitored using invasion chambers, and cell migration was analysed by scratch wound-healing assay. In vitro and ex vivo angiogenesis studies were performed by capillary-like tube formation assay and chick embryo angiogenesis assay (CEA). 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinoma in Sprague-Dawley rats. We observed a significant reduction in protein expression of vimentin, N-cadherin, Snail, Slug, Twist, MMP-2, MMP-9, p-EGFR, VEGFR-2, p-PI3K, Akt and p-GSK3β, and enhanced E-cadherin protein expression in response to AuNPs-Qu-5 treatment. AuNPs-Qu-5 inhibited migration and invasion of MCF-7 and MDA-MB-231 cells compared to free quercetin. AuNPs-Qu-5-treated HUVECs had reduced cell viability and capillary-like tube formation. In vitro and in vivo angiogenesis assays showed that AuNPs-Qu-5 suppressed tube and new blood vessel formation. Treatment with AuNPs-Qu-5 impeded tumour growth in DMBA-induced mammary carcinoma in SD rats compared to treatment with free quercetin. Our results suggest that AuNPs-Qu-5 inhibited EMT, angiogenesis and metastasis of the breast cancer cells tested by targeting the EGFR/VEGFR-2 signalling pathway.

Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry.

Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.

A mouse model to study the role of autophagy in obesity induced placental dysfunction

s / Placenta 35 (2014) A1eA112 A21 mediated via its receptors, and suggest that this phenomenon may play a role in situations of dysregulation of the vasoactive systems. P1.33. A MOUSE MODEL TO STUDY THE ROLE OF AUTOPHAGY IN OBESITY INDUCED PLACENTAL DYSFUNCTION Alina Maloyan, Sribalasubashini Muralimanoharan, Leslie Myatt University of Texas Health Science Center, San Antonio, Texas, USA Objectives: Maternal obesity leads to placental dysfunction and programs the offspring for metabolic disease. We have shown activation of oxidative/ nitrative stress, increase in p53 and mitochondrial dysfunction In placentas of obese women. Autophagy ensures cellular survival by sequestration of damaged organelles and proteins in autophagosomes, followed by degradation and recycling through the lysosomal machinery. We found disruption of autophagosome maturation in placentas of obese women leading to defective autophagy. To investigate the role of autophagy in placental function, a conditional knockout mouse model was generated with placenta-specific deletion of autophagy-related gene 7 (atg7). We have previously reported that deletion of ATG7 in placenta programmed the offspring to obesity and hyperglycemia. Here we hypothesized that ATG7 deficiency alters placental oxidative stress, cell death and mitochondrial function. Methods: Placentas from ATG7 knockout (KO, n1⁄45 dams) and wild type (WT, n1⁄45 dams) mice were collected at C-section on day 17.5 of gestation. Markers of oxidative stress, mitochondrial metabolism and cell death were measured by Western Blot. Result: When compared to WT mice, the KO mice showed a significant reduction in protein expression of subunits of mitochondrial complexes II, III and V as well as in expression of antioxidants SOD2 and catalase (p<0.05) indicating mitochondrial dysfunction and a decrease in oxidative stress defences. In the KO mouse placenta there was a significant increase in expression of both PPARg, a key regulator of mitochondrial metabolism in obesity (KO, 1.5±0.1 vs. WT, 0.9±0.0) and of p53 [KO, 1.3±0.1 vs. WT, 0.7±0.1). Apoptotic cell death was reduced in KO placentas as indicated by a 40% reduction in expression of both p53 upregulated modulator of apoptosis (PUMA) and of activated caspase-3 (p<0.05). Conclusion: Disruption of placental autophagic flux in the murine placenta leads to placental dysfunction, and fetal programming, similar to that in pregnancies with maternal obesity. P1.34-N. UPREGULATION OF PLACENTAL AUTOPHAGY IN A RAT MODEL OF DEXAMETHASONE-INDUCED FETAL GROWTH RESTRICTION Aisha Rasool, Ainslie Garrod, Elizabeth Cowley, Susan Greenwood, Elizabeth Cottrell University of Manchester, Manchester, UK Objectives: Exposure to stressors during pregnancy (e.g. poor nutrition or psychological stress) is associated with reduced fetal and placental growth. In animal models, the reduction in placental weight often precedes fetal growth restriction (FGR), suggesting placental adaptations support fetal growth in the face of an adverse environment. Autophagy, a conserved cellular process of ‘self-eating’ may be one mechanism by which this adaptation occurs, mobilizing placental energy stores to maintain fetal growth, at the expense of placental growth. Glucocorticoids (GCs) play an integral role in the response to in utero stressors, and in addition can stimulate autophagy in a number of non-placental cell types. Here, we sought to investigate the role of GCs in stimulating placental autophagy, using an established rat model of dexamethasone (DEX)-induced FGR. Methods: Pregnant rats were treated with dexamethasone acetate (DEX; 1ug/mL) or vehicle (0.1% ethanol) in drinking water from gestational day (G)13-G22. Fetal and placental weights were measured at G16, G19 and G22, and expression of autophagic genes assessed in placental tissue by qPCR. Result: Maternal DEX attenuated gestational weight gain (p<0.001), without affecting food intake. Placental weight was reduced by maternal DEX (by 14%, 28% and 34% compared with controls at G16, G19 and G22 respectively; p<0.001). Fetal weight was reduced to a lesser degree, at G19 and G22 (by 17.5% and 32.1%, respectively; p<0.05), indicating a degree of fetal protection from the growth-restricting effects of DEX. In the placenta, REDD1 and AMBRA1 gene expression were significantly increased by maternal DEX (p<0.01 for both), consistent with autophagic induction in this tissue. Conclusions: The current data suggest a role for glucocorticoids in the regulation of placental autophagy. Induction of autophagy in the placenta may underlie in part the ability of the placenta to support fetal growth in the face of increased stress or nutrient restriction. P1.35-N. SUPPLEMENTATION WITH INORGANIC NITRATE DURING PREGNANCY IMPROVES MATERNAL UTERINE ARTERY FUNCTION AND PLACENTAL EFFICIENCY IN MICE Elizabeth Cottrell , Ainslie Garrod , Mark Wareing , Mark Dilworth , Sarah Finn-Sell , SusanGreenwood , Philip Baker , Colin Sibley a University of Manchester, Manchester, UK; University of Auckland, Auckland, New

The Role Of BIN1 Tumor Suppressor Isoforms In Regulation Of Proliferation, Apoptosis and Tumor Formation Of Human Cutanous T-Cell Lymphoma Cells In Vitro and In Vivo

Cutaneous T-cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders that are characterized by homing of malignant T-cells to the surface of skin. There are two main types of CTCL: Mycosis Fungoides (MF) and its leukemic variant Sezary Syndrome (SS), which together represent about 65-70% of all CTCL cases. The precise genetic pathogenesis of these diseases remains largely undetermined. Recently, our research group has demonstrated that AHI-1 (Abelson Helper Integration site-1) oncogene is involved in CTCL. Expression of AHI-1 is increased in human leukemia cell lines, with marked upregulation (up to 40 fold) in CTCL lines (Hut78 and Hut102). Moreover, in FACS-purified CD4+CD7- Sezary cells from patients with Sezary Syndrome, AHI-1 expression is higher at both the RNA and protein levels compared to normal CD4+ cells. Furthermore, stable suppression of endogenous AHI-1 in Hut78 cells using small interfering RNA, normalizes their transforming activity both in vitro and in vivo. Thus, lymphomagenic activity of Hut78 cells is partially dependent on the expression of AHI-1. Interestingly, BIN1 (Bridging integrator 1) was identified through microarray analysis as one of the genes that may be involved in AHI-1-mediated leukemic transformation in CTCL cells. BIN1 is a nucleocytosolic adaptor protein with more than ten isoforms; some isoforms, including the BIN1 isoform (+10, +13), act as tumor suppressors, whereas the BIN1 (+12A) behaves as a cancer-related isoform in solid tumor models. However, the role of BIN1 in regulation of normal hematopoiesis and lymphomagenesis remains unknown. We have recently demonstrated that transcript levels of BIN1 isoforms are significantly lower in patients with MF or SS compared to controls. Four isoforms of BIN1 have been identified in Hut78 and primary CD4+CD7- Sezary cells. To investigate the role of BIN1 in CTCL, the BIN1 isoforms (+10, +13) and BIN1 (+12A) lentiviral constructs were transduced into two CTCL cell lines, Hut78 and HH cells. Overexpression of BIN1 isoforms led to a significant reduction in cell proliferation, as assessed by colony forming cell assays and 3H-Thymidine uptake assays (2-3 fold, p<0.05). Furthermore, a significant increase in spontaneous and specific apoptosis was observed in BIN1-transduced cells, with and without exogenous FAS-ligand (2-3 fold, p<0.05). Interestingly, a significant reduction in protein expression of c-FLIP (inhibitor of the FAS-mediated apoptosis pathway) and upregulation of downstream cleaved caspase-8 and caspase-3 was demonstrated in BIN1-transduced cells, suggesting that BIN1 isoforms induce apoptosis by downregulating the expression of c-FLIP, which leads to activation of the FAS-mediated apoptosis pathway. These findings show anti-proliferative and pro-apoptotic roles for BIN1 isoforms in human CTCL cells. In addition, subcellular fractionation and confocal microscopy further indicated that both the BIN1 (+10, +13) and BIN1 (+12A) isoforms are mainly located in the nucleus in Hut78 and HH cells. Furthermore, to investigate the effects of overexpression of BIN1 isoforms on the ability to induce tumors in vivo, we tested their leukemogenic potential by injecting transduced HH cells into non-obese diabetic/severe-combined immunodeficiency (NOD/SCID) mice. Mice injected subcutaneously with either parental HH or control empty vector cells (2 x 107/per mouse), showed local tumor formation in 6 of 6 mice within 4 days post-injection. The local tumors enlarged progressively and were often 1.5–2 cm in diameter by 3 weeks after injection. In contrast, no local tumors formed in mice given injections of equal numbers of BIN1-transduced HH cells after 14 days, in 12 out of 12 mice. Tumor formation was only observed in BIN1-transduced HH cells after 3 weeks post-injection. However, the local tumors were significantly smaller in BIN1-transduced HH cells compared to controls (∼4-fold). These findings further indicate that the two BIN1 isoforms have tumor suppressor activities in NOD/SCID mice and can significantly delay tumor formation and reduce tumor size in vivo. Disclosures:No relevant conflicts of interest to declare.

Abstract 9027: A Novel Polymorphism Arg146 of the Natriuretic Peptide Receptor-3 Gene Results in a Significant Reduction in Protein Expression Due to Accelerated Degradation

Background: The primary role of natriuretic peptide receptor 3 (NPR3) is clearance of natriuretic peptides. Genetic variation in NPR3 has been associated with blood pressure regulation and obesity. Despite its importance, variation in NPR3 characterizing coding single nucleotide polymorphisms (SNPs) has not been addressed by resequencing the gene. Objective: We set out to identify and functionally characterize common variation in NPR3 in 3 ethnic groups. DNA samples from such subjects are studied because common genetic variation needs to be defined initially in “normal” individuals prior to studying drug effect and response. Also, rare variants identified by sequencing are enriched in complex disease traits. Methods and Results: DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence NPR3. There were 105 polymorphisms identified of which 50 were novel, including 8 nonsynonymous (ns) SNPs of which 7 were novel. Expression constructs for the nsSNPs were created and HEK-293 cells were transfected with constructs for wild type (WT) and the variants. Recombinant proteins were analyzed by quantitative Western blot analysis. A significant reduction in NPR3 immunoreactive protein (60% or less of WT) was observed for the following variants Cys3, Arg146, Val499, and Ser477+Asp521. The Arg146 novel polymorphism resulted in the least protein expression (20% of WT). After treatment of WT and Arg146 cells with proteasome (MG132) and autophagy (3MA) inhibtors, it was determined that autophagy and not proteasomes plays an important role in the degradation of the Arg 146 variant protein. Modeling based on the NPR3 x-ray crystallography structure confirmed that the Arg146 variant were not compatible with WT conformation and could result in protein misfolding or instability. Conclusions: Multiple novel polymorphisms of NPR3 were identified in 3 ethnic groups. There were 4 variant receptors that displayed a significant decrease in NPR3 protein quantity, Arg146 being the variant with the least expression. Degradation of the variant Arg146 was mediated predominantly by autophagy. This SNP could have a significant effect on the metabolism of natriuretic peptides with clinical implications.

Epigenetic silencing of Id4 identifies a glioblastoma subgroup with a better prognosis as a consequence of an inhibition of angiogenesis

Inhibitors of DNA binding/differentiation (Id1 to Id4) are a family of helix-loop-helix transcription factors, which are highly expressed during embryogenesis and at lower levels in mature tissues. Id4 plays an important role in neuronal stem cell differentiation, and its deregulation has been implicated in glial neoplasia. The methylation status of Id4 was analyzed by methylation-specific polymerase chain reaction (PCR) in 62 glioblastoma (GBM) cases and in 20 normal brain tissues. Methylation status of Id4 was confirmed by sequencing after subcloning and messenger RNA (mRNA) and protein expression. We also evaluated the mRNA expression of MGP (matrix GLA protein), TGF-β1 (transforming growth factor beta 1), and VEGF (vascular endothelial growth factor) by real-time PCR analysis. Clinical and histological assessment of tumor angiogenesis was performed by evaluating the relative enhancing tumor ratio on magnetic resonance imaging and microvessel density on von Willebrand factor-stained sections, respectively. The promoter of Id4 was methylated in 23 of 62 (37%) GBMs. In methylated GBMs, Id4 mRNA was significantly reduced, compared with unmethylated GBMs (P = .0002). A significant reduction of protein expression was detected in all hypermethylated cases. GBMs with methylated Id4 showed a significant reduction of MGP, TGF-β1, and VEGF mRNA expression and had significantly lower relative enhancing tumor ratio (P = .0108) and microvessel density (P = .0241) values with respect to unmethylated GBMs. Finally, Id4 methylation was significantly associated with a favorable clinical outcome (P = .0006). These data suggest that methylation of Id4 may be involved in the pathogenesis of GBM and in the resistance of this neoplasm to conventional treatment throughout MGP-mediated neoangiogenesis.

Prognostic relevance of promoter methylation-associated loss of ID4 expression in glioblastoma multiforme.

2076 Background: Inhibitors of DNA binding/differentiation (ID1-4) are a family of helix-loop-helix transcription factor, highly expressed during embryogenesis and at lower levels in mature tissues. Expression of ID proteins have related to features of malignancies, including cellular transformation, angiogenesis and metastasis. ID4 plays an important role in neuronal stem cell differentiation and its deregulation has been implicated in glial neoplasia. Recent works have shown that hypermethylation of ID4 can determine the silencing of this protein in several human cancers. Methods: We analyzed the methylation status of ID4 by methylation-specific-PCR in 85 glioblastoma multiforme (GBM) cases and in 20 normal brain tissues. After surgical treatment, all GBMs were subjected to a cycle of radiotherapy (2 Gy per fraction, once a day, 5 days a week, 60 Gy total dose) with concomitant administration of temozolomide (75 mg per square meter of body surface area per day) for 7 days a week from the first to the last day of radiotherapy, followed by 5 cycles of adjuvant temozolomide (at 200 mg per square meter of body surface area on days 1 through 5) given at four-week intervals. Methylation status was confirmed by sequencing after subcloning and RNA and protein expression was assessed by real-time PCR and immunohistochemistry. The relationship between ID4 methylation and clinical outcome was investigated. Results: The promoter of ID4 was methylated in 17/85 GBMs. After subcloning, the methylation was confirmed by sequencing in over 95% of GpC dinucleotides. In methylated GBMs, ID4 mRNA expression was reduced by 5 folds as compared with unmethylated GBMs. A significant reduction of protein expression was detected in all hypermethylated cases. ID4 methylation was significantly associated with a favourable clinical outcome (p=0.0033). No significant differences were found between patients with and without ID4 methylation with respect to age, sex, p53 mutation, Ki67 labeling index and extent of surgical resection. Conclusions: Our data suggest that methylation of ID4 may be involved in the pathogenesis of GBM and in the resistance of this neoplasm to conventional treatment.

Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

BackgroundModern RNA interference (RNAi) methodologies using small interfering RNA (siRNA) oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules.Principal FindingsHere we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA) targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica β-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite.ConclusionsOur results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

Pleiotrophin and Transgelin, Two Proteins Important in Cell Migration, Are Targeted by Non‐Steroidal Anti‐Inflammatory Drugs

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are among the most widely used drugs today. Chronic treatment with NSAIDs can result in gastrointestinal (GI) toxicity. Microarray analysis of intestinal epithelial cell (IEC‐6) monolayers revealed that mRNA expression of transgelin (TAGLN), an actin binding protein of the calponin family, and pleiotrophin (PTN), a heparin binding protein involved in angiogenesis, are suppressed by NSAID‐treatment. qRT‐PCR and sequence analysis confirmed that PTN and TAGLN messages are down significantly in the presence of indomethacin and NS‐398, NSAIDs that inhibit IEC‐6 migration, but not SC‐560, an NSAID that does not inhibit migration. NSAIDs also suppress protein expression of TAGLN and PTN in IEC‐6 cells. Additionally, TAGLN mRNA expression is up‐regulated in migrating cells versus non‐migrating cells. Furthermore, there was a significant reduction in protein expression of PTN from mucosa of NSAID‐treated equine stomach, small intestine, cecum, and large intestine. Our results indicate that expression of PTN and TAGLN are down‐regulated by NSAIDs. Thus, PTN and TAGLN may have an important role in cell migration, and NSAID‐induced suppression of PTN and TAGLN expression may account for at least a portion of the inhibition of cell migration caused by NSAIDs in IEC‐6 cells. Finally, reduced PTN may play a role in the clinical development of NSAID induce GI ulceration.

Resolution of endothelial activation and down-regulation of Tie2 receptor in psoriatic skin after infliximab therapy

Psoriasis is a common dermatosis characterized by erythematous skin plaques and associated arthritis. Microvessels of the papillary dermis in psoriatic lesions are elongated, tortuous, and dilated, which contributes significantly to the proinflammatory response. Angiopoietin (Ang) 1 and 2 and their receptor, Tie2, are a family of growth factors recognized in inflammatory lesions to be critical for new blood vessel growth and maintenance, with recent studies suggesting tumor necrosis factor (TNF)-alpha-induced angiogenesis is in part mediated by the Tie2 receptor. The aim of this study was to evaluate the effect of anti-TNF-alpha therapy on angiogenic growth factor expression and on the cellular infiltrate in psoriatic lesional skin. Sixteen patients with moderate to severe psoriasis and associated psoriatic arthritis (n = 13) received infliximab infusions (3-5 mg/kg) at baseline and at 2 and 6 weeks. Clinical assessments and skin biopsies were undertaken at baseline, and at 2 and 12 weeks. Ang 1, Ang 2, Tie2, and TNF-alpha messenger RNA expression were quantified by real-time polymerase chain reaction. Protein expression of vascular endothelial growth factor, Ang 2, Tie2, TNF-alpha, and the inflammatory infiltrate was determined using immunohistology. We conducted clinical assessments including Psoriasis Area and Severity Index, percentage body surface area, Arthritis Disease Activity Score, and Health Assessment Questionnaire. At baseline expression of Ang 1/2, vascular endothelial growth factor, Tie2, and TNF-alpha messenger RNA and protein were greater in preinvolved skin compared with uninvolved skin (P < .05). Infliximab produced a significant reduction in protein expression of Ang 2, vascular endothelial growth factor, and Tie2 (P < .001) along with a decrease in messenger RNA expression of Ang 1 (P < .045) and Tie2 (P < .021). This was paralleled by a significant reduction in the inflammatory infiltrate scores (P < .001) and platelet-endothelial cell adhesion molecule (CD31) expression (P = .001), suggesting deactivation of endothelial cell. There was a 93% mean reduction in Psoriasis Area and Severity Index (P = .001), and a significant reduction in Disease Activity Score 28 (P = .012) and mean Health Assessment Questionnaire scores by week 12. This study involves a small number of patients. These results suggest infliximab is both effective and well tolerated in severe psoriasis, resulting in deactivation of endothelium and down-regulation of growth factor and cytokine expression, leading to a decrease in the cellular infiltrate and clinical improvement in psoriasis. Furthermore, the effect of infliximab on growth factor expression, in particular Tie2, supports previous in vitro work suggesting TNF-alpha may be a major regulator of the Ang/Tie2 pathway.

The Role of the Leader Sequence Coding Region in Expression and Assembly of Bacteriorhodopsin

Bacterio-opsin is made as a precursor in Halobacterium halobium, which has 13 additional residues at the amino terminus. The codons for these residues have been proposed to form a hairpin structure in the mRNA and play a role in ribosome binding; the leader peptide sequence also has been proposed to have a role in membrane insertion of bacteriorhodopsin (BR). We have made mutations in the bop gene region coding for the leader sequence and expressed the mutant genes in an H. halobium mutant lacking wild-type BR. The leader sequence coding region was found to be important for the stability of the mRNA and for its efficient translation. Single base substitutions in this region that did not affect the amino acid sequence caused significant reductions in protein expression. Deletion of the leader region resulted in unstable mRNA and almost no BR production. Introduction of a new ribosome-binding sequence within the coding region of the mature protein restored mRNA stability and some protein expression. Protein made without the leader peptide was properly assembled in the membrane.