Accelerated Aging Stability of ß-Ga2O3-Titanium/Gold Ohmic Interfaces.

Stable ohmic contacts are critical to enable efficient operation of high-voltage electronic devices using ultrawide bandgap semiconductors. Here we perform, for the first time, thermally accelerated aging of Ti/Au ohmic interfaces to (010) ß-Ga2O3. We find that a heavily doped semiconductor, doped n-type by Si-ion implantation, treated with reactive ion etch (RIE), results in a low specific contact resistance of ∼10-5 Ω cm2 that is stable upon accelerated thermal aging at 300 °C for 108 h. The low resistance interface is due to thermionic field emission of electrons over an inhomogeneous barrier. Scanning/transmission electron microscopy indicates that the multi-layered structure and elemental distribution across the contact interface, formed during a 1 min 470 °C post-metallization anneal, do not change noticeably over the aging period. A ∼1 nm interfacial layer is observed by high-resolution microscopy at the Ti-TiOx/Ga2O3 interface on all samples exposed to RIE, which may contribute to their excellent stability. In addition, longer-range facet-like interfacial features are observed, which may contribute to the inhomogeneous barrier. In contrast, Ti/Au junctions to moderately doped (010) Ga2O3 made with no RIE treatment exhibit a high contact resistance that increases upon accelerated aging, along with a partially lattice-matched interface. The methods used here to understand the process, structure, and electrical property relationships for Ti/Au contact interfaces to ß-Ga2O3 can be applied to assess and tune the stability of a variety of other oxide-semiconductor interfaces.

Marked variability in bioactivity between commercially available bovine colostrum for human use; implications for clinical trials.

BACKGROUND: Colostrum, the milk produced during first few days after birth, is rich in immunoglobulins, antimicrobial peptides & growth factors. Multiple clinical trials using bovine colostrum are ongoing but with no assessment of test product bioactivity. OBJECTIVES: To examine variability of bioactivity between 20 commercial colostrum products, contribution of TGFß and EGFR in mediating effects, heat sensitivity of bioactivity and changes in bioactivity of colostrum milkings in the days following calving. DESIGN: In vitro bioactivity used AGS, RIE-1 and Caco-2 cell proliferation (Alamar blue) and migration (wounded monolayers) assays. Changes in colostrum bioactivity determined following addition of TGFß-neutralising antibody, EGFR blocker (Typhostin) and after heating (40-60°C, 60 min). In vivo bioassay assessed ability of colostrum gavage (2ml, 7mg/ml) to reduce gastric damage (NSAID + restraint) in rats. Milkings from 6 cows, days 0-3 post calving were assessed for bioactivity and growth factor concentrations. RESULT: Six-fold differences in pro-proliferative and migratory activity were seen comparing commercial products. Comparison of most- and least-active samples from in vitro studies showed two- to three-fold differences in ability to reduce gastric injury (86% reduction using most-active vs 48% using least-active, p<0.01). Tyrphostin reduced pro-migratory and proliferative activity by 23% and 55%. TGFß neutralisation reduced migratory activity by 83% but did not affect proliferation Heating colostrum powder to 50°C did not affect immunoactivity of haptoglobin, EGF, TGFß, IgG, IGF-1 or betacellulin but decreased bioactivity by >40%. Milking studies showed high bioactivity during first and second milkings on day 0 but 77% reduction by day 3. Changes in total protein, haptoglobin, EGF, TGFß, IgG and IGF-1 paralleled falls in bioactivity. CONCLUSION: Commercial colostrum products possess widely different bioactivity. Variation in heat exposure and/or proportion of day 0 colostrum content may contribute to this. Assessment of colostrum bioactivity has advantages to growth factor quantitation for quality control.

Experiência de c rie e doen‡a periodontal em crian‡as e adolescentes com paralisia cerebral / Dental caries and periodontal disease in children and adolescents with cerebral palsy

O objetivo deste estudo foi identificar os fatores associados … experiˆncia de c rie e doen‡a periodontal em crian‡as e adolescentes com paralisia cerebral (PC). Foram inclu¡dos neste estudo 70 crian‡as e adolescentes com PC, de ambos os sexos, na faixa et ria de 6 a 19 anos de idade. As vari veis estudadas foram idade, sexo, escolaridade, classifica‡ães da PC, GMFCS, uso de medicamentos, motricidade oral e consistˆncia da dieta. A experiˆncia de c rie foi avaliada pelo ¡ndice CPO-D, as condi‡ães periodontais pelo ¡ndice de higiene oral simplificado (IHO-S) e sangramento gengival … sondagem. Os exames epidemiol¢gicos foram realizados por uma £nica examinadora calibrada (Kappa = 0,97). Foram realizadas an lises univariada e multivariada, com n¡vel de significƒncia de 5%. A maioria dos participantes deste estudo era do sexo masculino (n=40, 57,1%), com PC do tipo esp stica (n=56, 80%), tetrapl‚gicos (n=50, 71,4%), GMFCS n¡vel V (n=41, 58,5%), com motricidade oral funcional (n=28, 40%). Faziam uso de anticonvulsivantes (n=31, 44,2%) e consumiam dieta s¢lida (n=38, 54,2%). O ¡ndice m‚dio de CPO-D = 3,8, sendo que a maioria dos participantes apresentava CPO-D=0 (n=45, 64,3%) e a presen‡a de sangramento gengival observada em 48 dos avaliados foi de 68,6%. O aumento da idade e a ingestÆo de dieta semi-s¢lida aumentam as chances desses indiv¡duos apresentarem maiores valores de experiˆncia de c rie. A presen‡a de maior comprometimento motor expresso pelo GMFCS foi n¡vel V, motricidade oral semifuncional e uso de anticonvulsivantes aumentam a frequˆncia de sangramento gengival em crian‡as e adolescentes com PC.

The aim of this study was to identify factors associated with dental caries and periodontal disease in children and adolescents with CP. The study included 70 children and adolescents with CP, of both sexes aged from 6 to 19 years old. The studied variables were age, sex, education, and ratings of PC, GMFCS, medications, oral motor, and consistency of the diet. Caries were assessed by the decayed, missing and filled teeth (DMFT); and periodontal conditions by the simplified oral hygiene index (OHI-S) and bleeding on probing. The epidemiological exam was performed by a single calibrated researcher (kappa = .97). We performed univariate and multivariate analyzes, with a significance level of 5%. Most participants of the study were male (n = 40, 57.1%), with CP spastic type (n = 56, 80%), tetraplegia (n = 50, 71.4%), GMFCS level V (n = 41, 58.5%) with oral motor function (n = 28, 40%). Patients of anticonvulsants (n = 31, 44.2%) and were on a solid diet (n = 38, 54.2%). The average DMFT = 3.8, the majority of the patients t had DMFT = 0 (n = 45, 64.3%) gingival bleeding was observed in 48 patients (68.6%). Increasing age and intake of semisolid increase the chances of these individuals presenting higher values of caries experience. Greater motor impairment expressed by GMFCS was level V, oral motor semi-functional, and anticonvulsants increase the frequency of gingival bleeding in children and adolescents with CP.

Rubus idaeus L. reverses epithelial-to-mesenchymal transition and suppresses cell invasion and protease activities by targeting ERK1/2 and FAK pathways in human lung cancer cells.

Epithelial to mesenchymal transition (EMT) has been considered essential for cancer metastasis, a multistep complicated process including local invasion, intravasation, extravasation, and proliferation at distant sites. Herein we provided molecular evidence associated with the antimetastatic effect of Rubus idaeus L. extracts (RIE) by showing a nearly complete inhibition on the invasion (p<0.001) of highly metastatic A549 cells via reduced activities of matrix metalloproteinase-2 (MMP-2) and urokinasetype plasminogen activator (u-PA). We performed Western blot to find that RIE could induce up-regulation of epithelial marker such as E-cadherin and α-catenin and inhibit the mesenchymal markers such as N-cadherin, fibronectin, snail-1, and vimentin. Selective snail-1 inhibition by snail-1-specific-siRNA also showed increased E-cadherin expression in A549 cells suggesting a possible involvement of snail-1 inhibition in RIE-caused increase in E-cadherin level. RIE also inhibited p-FAK, p-paxillin and AP-1 by Western blot analysis, indicating the anti-EMT effect of RIE in human lung carcinoma. Importantly, an in vivo BALB/c nude mice xenograft model showed that RIE treatment reduced tumor growth by oral gavage, and RIE represent promising candidates for future phytochemical-based mechanistic pathway-targeted cancer prevention strategies.

Lack of functionally active sweet taste receptors in the jejunum in vivo in the rat.

BACKGROUND: When studied in enterocyte-like cell lines (Caco-2 and RIE cells), agonists and antagonists of the sweet taste receptor (STR) augment and decrease glucose uptake, respectively. We hypothesize that exposure to STR agonists and antagonists in vivo will augment glucose absorption in the rat. MATERIALS AND METHODS: About 30-cm segments of jejunum in anesthetized rats were perfused with iso-osmolar solutions containing 10, 35, and 100 mM glucose solutions (n = 6 rats, each group) with and without the STR agonist 2 mM acesulfame potassium and the STR inhibitor 10 µM U-73122 (inhibitor of the phospholipase C pathway). Carrier-mediated absorption of glucose was calculated by using stereospecific and nonstereospecific (14)C-d-glucose and (3)H-l-glucose, respectively. RESULTS: Addition of the STR agonist acesulfame potassium to the 10, 35, and 100 mM glucose solutions had no substantive effects on glucose absorption from 2.1 ± 0.2 to 2.0 ± 0.3, 5.8 ± 0.2 to 4.8 ± 0.2, and 15.5 ± 2.3 to 15.7 ± 2.7 µmoL/min/30-cm intestinal segment (P > 0.05), respectively. Addition of the STR inhibitor (U-73122) also had no effect on absorption in the 10, 35, and 100 mM solutions from 2.3 ± 0.1 to 2.1 ± 0.2, 7.7 ± 0.5 to 7.2 ± 0.5, and 15.7 ± 0.9 to 15.2 ± 1.1 µmoL/min/30-cm intestinal segment, respectively. CONCLUSIONS: Provision of glucose directly into rat jejunum does not augment glucose absorption via STR-mediated mechanisms within the jejunum in the rat. Our experiments show either no major role of STRs in mediating postprandial augmentation of glucose absorption or that proximal gastrointestinal tract stimulation of STR or other luminal factors may be required for absorption of glucose to be augmented by STR.

Effect of the artificial sweetener, acesulfame potassium, a sweet taste receptor agonist, on glucose uptake in small intestinal cell lines.

INTRODUCTION: Sweet taste receptors may enhance glucose absorption. AIM: This study aimed to explore the cell biology of sweet taste receptors on glucose uptake. HYPOTHESIS: Artificial sweeteners increase glucose uptake via activating sweet taste receptors in the enterocyte to translocate GLUT2 to the apical membrane through the PLC ßII pathway. METHODS: Caco-2, RIE-1, and IEC-6 cells, starved from glucose for 1 h were pre-incubated with 10 mM acesulfame potassium (AceK). Glucose uptake was measured by incubating cells for 1 to 10 min with 0.5-50 mM glucose with or without U-73122, chelerythrine, and cytochalasin B. RESULTS: In Caco-2 and RIE-1 cells, 10 mM AceK increased glucose uptake by 20-30 % at glucose >25 mM, but not in lesser glucose concentrations (<10 mM), nor at 1 min or 10 min incubations. U-73122 (PLC ßII inhibitor) inhibited uptake at glucose >25 mM and for 5 min incubation; chelerythrine and cytochalasin B had similar effects. No effect occurred in IEC-6 cells. Activation of sweet taste receptors had no effect on glucose uptake in low (<25 mM) glucose concentrations but increased uptake at greater concentrations (>25 mM). CONCLUSIONS: Role of artificial sweeteners on glucose uptake appears to act in part by effects on the enterocyte itself.

Concurrent recordings of bladder afferents from multiple nerves using a microfabricated PDMS microchannel electrode array.

In this paper we present a compliant neural interface designed to record bladder afferent activity. We developed the implant's microfabrication process using multiple layers of silicone rubber and thin metal so that a gold microelectrode array is embedded within four parallel polydimethylsiloxane (PDMS) microchannels (5 mm long, 100 µm wide, 100 µm deep). Electrode impedance at 1 kHz was optimized using a reactive ion etching (RIE) step, which increased the porosity of the electrode surface. The electrodes did not deteriorate after a 3 month immersion in phosphate buffered saline (PBS) at 37 °C. Due to the unique microscopic topography of the metal film on PDMS, the electrodes are extremely compliant and can withstand handling during implantation (twisting and bending) without electrical failure. The device was transplanted acutely to anaesthetized rats, and strands of the dorsal branch of roots L6 and S1 were surgically teased and inserted in three microchannels under saline immersion to allow for simultaneous in vivo recordings in an acute setting. We utilized a tripole electrode configuration to maintain background noise low and improve the signal to noise ratio. The device could distinguish two types of afferent nerve activity related to increasing bladder filling and contraction. To our knowledge, this is the first report of multichannel recordings of bladder afferent activity.

Antiviral Decoction of Isatidis Radix ( bǎn lán gen) Inhibited Influenza Virus Adsorption on MDCK Cells by Cytoprotective Activity.

The aim of this study is to elucidate how the Isatidis Radix ( bǎn lán gen) tonic, as an aqueous mixture of hundreds of compositions, interrupts the infection of influenza viruses to their host cells. The efficacy of the tonic was evaluated and expressed as cell proliferation rate and plaque reduction rate in Madin-Darby Canine Kidney (MDCK) cells, against 3 strains of influenza A and B viruses. This boiling water (at 100°C) extract of Isatidis Radix (RIE) showed antiviral activity against influenza virus A and B. The concentration for 50% inhibition of influenza virus A replication (IC50) in MDCK cell was 12.6 mg/mL with a therapeutic index >8. When cells were incubated with RIE prior to virus adsorption, the numbers of viable cell were at least doubled compared to the numbers of virus control, RIE incubation after virus adsorption and RIE incubation with virus prior to adsorption, in both influenza virus A and B. Moreover, much less virus particles were spotted by scanning electron microscope (SEM) in the RIE pre-treated cells than the cells without RIE treatment. These results indicate the antiviral activity of RIE is mainly attributed to its host cell protection effect but not actions on virus or post-virus-adsorption interruption. Cell, but not virus, is more likely to be the action target of RIE.

Mechanisms of glucose uptake in intestinal cell lines: role of GLUT2.

BACKGROUND: GLUT2 is translocated to the apical membrane of enterocytes exposed to glucose concentrations >∼50 mM. Mechanisms of GLUT2-mediated glucose uptake in cell culture models of enterocytes have not been studied. AIM: To explore mechanism(s) of glucose uptake in 3 enterocyte-like cell lines. METHODS: Glucose uptake was measured in Caco-2, RIE-1, and IEC-6 cell lines using varying concentrations of glucose (0.5-50 mM). Effects of phlorizin (SGLT1 inhibitor), phloretin (GLUT2 inhibitor), nocodazole and cytochalasin B (disrupters of cytoskeleton), calphostin C and chelerythrine (PKC inhibitors), and phorbol 12-myristate 13-acetate (PKC activator) were evaluated. RESULTS: Phlorizin inhibited glucose uptake in all 3 cell lines. Phloretin inhibited glucose uptake in Caco-2 and RIE-1 cells. Starving cells decreased glucose uptake in Caco-2 and RIE-1 cells. Glucose uptake was saturated at >10 mM glucose in all 3 cell lines when exposed briefly (<1 min) to glucose. After exposure for >5 min in Caco-2 and RIE-1 cells, glucose uptake did not saturate and K(m) and V(max) increased. This increase in glucose uptake was inhibited by phloretin, nocodazole, cytochalasin B, calphostin C, and chelerythrine. PMA enhanced glucose uptake by 20%. Inhibitors and PMA had little or no effect in the IEC-6 cells. CONCLUSION: Constitutive expression of GLUT2 in the apical membrane along with additional translocation of cytoplasmic GLUT2 to the apical membrane via an intact cytoskeleton and activated PKC appears responsible for enhanced carrier-mediated glucose uptake at greater glucose concentrations (>20 mM) in Caco-2 and RIE-1 cells. IEC-6 cells do not appear to express functional GLUT2.

Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells.

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3'UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3'UTR that may act as regulatory sequences. Analysis of ODC 3'UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

Pattern of presentation of oral health conditions among children at the University of Port Harcourt Teaching Hospital (UPTH), Port Harcourt, Nigeria

Objective: To evaluate the spectrum of oral health conditions that presented at the Child Dental Health Clinic. Methods: A 30-month retrospective study of oral health conditions of 462 children, 222 (48.3%) males and 238 (51.7%) females, aged 16 years and below seen at the Dental Centre, University of Port Harcourt Teaching Hospital, Port Harcourt, Nigeria. Results: The average age of the patients was 8.5 + 3.8 (SD) years. Nineteen (4.1%) children attended for routine dental check-up and there were no routine visits before the age of one year. Two hundred and fifteen (46.2%) of the children were diagnosed with dental caries, while 91 (19.7%) of the children had chronic gingivitis. Only 42 (9.1%) children attended due to traumatic injuries to the dentition. Forty six (10%) were referred for orthodontic management. There was a female predilection for dental caries (58.1%) while the males were found to have more periodontal diseases. Conclusion: There is a poor dental awareness as reflected by the proportion of children that attended the clinic for routine visits and the severity of some of the dental conditions suggesting late presentations. An early age dental visit should be encouraged since it provides a foundation upon which a lifetime of preventive education and oral health care can be developed.

Objetivo: Avaliar as condições de saúde bucal de crianças atendidas na Clínica Odontológica Infantil. Método: Um estudo restrospectivo de 30 meses foi conduzido em 462 crianças - 222 (48,3%) do sexo masculino e 238 (51,7%) do sexo feminino, com at‚ 16 anos de idade atendidas no Centro de Odontologia da Universidade de Port Harcourt Teaching Hospital, Port Harcourt, na Nig‚éia. Resultados: A idade média dos pacientes foi de 8,5+3,8 anos. Dezenove (4,1%) crian‡çs foram atendidas para exame odontológico de rotina, não existindo consultas odontológicas nates de um ano de idade. Duzentos e quinze crianças (46,2%) possuiam lesães de c rie dent ria, enquanto 91 (19,7%) apresentavam gengivite crônica. Apenas 42 crian‡as (9,1%) foram atendidas devido a traumatismos dentários. Quarenta e seis crianças (10,0%) foram encaminhadas para tratamento ortodôntico. Houve uma predileção do sexo feminino para a cárie dentária (58,1%), enquanto os meninos apresentaram maior frequência de doença periodontal. Conclusão: H  uma deficiência nos cuidados dentários que‚ refletido pela proporção de crianças que compareceram ao ambulatório para visitas de rotina e pela gravidade de algumas das condições dentárias apresentadas sugerindo uma procura tardia pelos serviços de saúde. A consulta odontológica precoce deve ser incentivada, pois proporciona uma base sobre a qual a educação preventiva em sa£de e cuidados de saúde bucal podem ser implementados.

Dietary fiber enhances TGF-ß signaling and growth inhibition in the gut.

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-ß signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-ß represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-ß's growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-ß(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-ß-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-ß alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-ß synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-ß induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.

Reactive oxygen species-quenching and anti-apoptotic effect of polaprezinc on indomethacin-induced small intestinal epithelial cell injury.

BACKGROUND: To protect the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs is one of the critical issues in the field of gastroenterology. Polaprezinc (PZ), a gastric muco-protecting agent, has been widely used for the treatment of gastric ulcer and gastritis for its unique effects, such as its strong reactive oxygen species (ROS)-quenching effect. The aim of this study was to clarify the mechanism by which indomethacin-induced small intestinal mucosal injury occurs, by using a rat intestinal epithelial cell line (RIE-1). In addition, the protective role of PZ and the possible mechanism of its effect on indomethacin-induced small intestinal injury were investigated. METHODS: Cell death was evaluated by methyl thiazolyl tetrazolium (MTT) assay and a double-staining method with Hoechst33342 dye and propidium iodide. Indomethacin-induced ROS production was evaluated by detecting the oxidation of a redox-sensitive fluorogenic probe, RedoxSensor, and the oxidation of cysteine residues of proteins (protein S oxidation). The activation of cytochrome c, smac/DIABLO, and caspase-3 was assessed by western blotting. In some experiments, PZ or its components, L: -carnosine and zinc, were used. RESULTS: We found that indomethacin caused apoptosis in RIE-1 cells in a dose- and time-dependent manner. Indomethacin also induced ROS production and an increase in the protein S oxidation of RIE-1. Pretreatment of RIE-1 with PZ or zinc sulfate, but not L: -carnosine, significantly reduced the indomethacin-induced apoptosis. PZ prevented ROS production and the increase in protein S-oxidation. PZ inhibited indomethacin-induced cytochrome c and smac/DIABLO release and subsequent caspase-3 activation. CONCLUSIONS: The protective effect of PZ on indomethacin-induced small intestinal injury may be dependent on its ROS-quenching effect.

PKD prevents H2O2-induced apoptosis via NF-kappaB and p38 MAPK in RIE-1 cells.

Previously, we demonstrated that protein kinase D (PKD) plays a protective role during H(2)O(2)-induced intestinal cell death. Here, we sought to determine whether this effect is mediated by nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPKs). Treatment with H(2)O(2) activated NF-kappaB in RIE-1 cells; H(2)O(2) also induced the translocation of NF-kappaB p65 as well as phosphorylation of IkappaB-alpha. PKD1 siRNA inhibited H(2)O(2)-induced activation, translocation of NF-kappaB, and phosphorylation of IkappaB-alpha. We also found that overexpression of wild type PKD1 attenuated H(2)O(2)-induced phosphorylation of p38 MAPK and its upstream activator, MAPK kinase (MKK) 3/6, whereas the phosphorylation was increased by PKD1 siRNA or kinase-dead PKD1. Phosphorylation of neither extracellular signal-regulated kinases (ERK) 1/2 nor c-Jun N-terminal kinases (JNK) was altered by PKD1 plasmids or siRNA. Our findings suggest that PKD protects intestinal cells through up-regulation of NF-kappaB and down-regulation of p38 MAPK.

Peroxisome proliferator-activated receptor gamma down-regulates follistatin in intestinal epithelial cells through SP1.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) down-regulates the expression of follistatin mRNA in intestinal epithelial cells in vivo. The mechanism of PPARgamma-mediated down-regulation of follistatin was investigated using non-transformed, rat intestinal epithelial cells (RIE-1). RIE cells expressed activin A, the activin receptors ActRI and ActRII, and the follistatin-315 mRNA. RIE-1 cells responded to endogenous activin A, and this response was antagonized by follistatin, as evidenced by changes in cell growth and regulation of an activin-responsive reporter. Using RIE-1 cells, we show that activation of PPARgamma by rosiglitazone reduced follistatin mRNA levels in a dose- and concentration-dependent manner. Down-regulation of follistatin by rosiglitazone required the DNA binding domain of PPARgamma and was dependent upon dimerization with the retinoid X receptor. Inhibition of follistatin expression by rosiglitazone was not associated with decreased follistatin mRNA stability, suggesting that regulation may be at the promoter level. Analysis of the follistatin promoter revealed consensus binding sites for AP-1, AP-2, and Sp1. Targeting the AP-1 pathway with SP600125, an inhibitor of JNK, and TAM67, a dominant negative c-Jun, had no effect on PPARgamma-mediated down-regulation of follistatin. However, the follistatin promoter was dramatically regulated by Sp1, and this regulation was inhibited by PPARgamma expression. Knockdown of Sp1 expression relieved repression of follistatin levels by rosiglitazone. Moreover, PPARgamma was found to interact with Sp1 and repress its transcriptional activation function. Collectively, our data indicate that repression of Sp1 transcriptional activity by PPARgamma is the underlying mechanism responsible for PPARgamma-mediated regulation of follistatin expression.

Transformation by oncogenic Ras expands the early genomic response to transforming growth factor beta in intestinal epithelial cells.

A substantial body of evidence implicates TGFbeta as a tumor promoter in epithelial cells that have become resistant to its tumor suppressor activity. To better understand early, genome-wide TGFbeta responses in cells resistant to growth inhibition by TGFbeta, we used microarray analysis in a well-defined cell culture system of sensitive and resistant intestinal epithelial cells. TGFbeta-regulated gene expression in TGFbeta-growth-sensitive, nontransformed rat intestinal epithelial cells (RIE-1) was compared to expression in TGFbeta-growth-resistant RIE cells stably transformed by oncogenic Ras(12V). Treatment of RIE-1 cells with 2 ng/ml TGFbeta1 for 1 hour increased the expression of eight gene sequences by 2.6-fold or more, whereas eight were down regulated 2.6-fold. In RIE-Ras(12V) cells, 42 gene sequences were upregulated and only 3 were down-regulated. Comparison of RIE and RIE-Ras(12V) identified 37 gene sequences as unique, Ras-dependent genomic targets of TGFbeta1. TGFbeta-regulation of connective tissue growth factor and vascular endothelial growth factor, two genes up-regulated in RIE-Ras cells and previously implicated in tumor promotion, was independently confirmed and further characterized by Northern analysis. Our data indicate that overexpression of oncogenic Ras in intestinal epithelial cells confers a significantly expanded repertoire of robust, early transcriptional responses to TGFbeta via signaling pathways yet to be fully elucidated but including the canonical Raf-1/MAPK/Erk pathway. Loss of sensitivity to growth inhibition by TGFbeta does not abrogate TGFbeta signaling and actually expands the early transcriptional response to TGFbeta1. Expression of some of these genes may confer to Ras-transformed cells characteristics favorable for tumor promotion.

Frequent overexpression of Aurora Kinase A in upper gastrointestinal adenocarcinomas correlates with potent antiapoptotic functions.

BACKGROUND: Upper gastrointestinal adenocarcinomas are a common cause of cancer-related deaths. In this study, the authors investigated the prevalence and biological significance of Aurora Kinase A (AURKA) overexpression in upper gastrointestinal adenocarcinomas. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining on tumor tissue microarrays (TMA) were used to study the expression of AURKA in upper gastrointestinal adenocarcinomas. To investigate the biological and signaling impact of AURKA, the authors used multiple in vitro assays that included 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling), cytochrome C release, flow cytometry, luciferase reporter, and Western blot analysis. RESULTS: Frequent overexpression of AURKA transcript in upper gastrointestinal adenocarcinomas was detected compared with normal samples (47%; P= .001). The immunohistochemical analysis of 130 tumors demonstrated moderate-to-strong immunostaining of AURKA in >50% of upper gastrointestinal adenocarcinomas. By using camptothecin as a drug-induced apoptosis in vitro model, the authors demonstrated that the expression of AURKA provided protection against apoptosis to gastrointestinal cancer cells (AGS and RKO) (P= .006) and RIE-1 primary intestinal epithelial cells (P= .001). The AURKA overexpression mediated an increase in phosphorylation of AKT(Ser473) with an increase in HDM2 level. The shRNA-knockdown of AKT in AURKA-overexpressing cells reversed this effect and showed a significant increase in the p53 protein level, indicating a possible nexus of AURKA/AKT/p53. Indeed, overexpression of AURKA led to a remarkable reduction in the transcription activity of p53, with subsequent reductions in transcript and protein levels of its downstream proapoptotic transcription targets (p21, BAX, NOXA, and PUMA). CONCLUSIONS: Study results indicated that AURKA provides potent antiapoptotic properties to gastrointestinal cells by regulating levels of p53 through the AKT/HDM2 axis.

Synergistic effect between EGF and TGF-beta1 in inducing oncogenic properties of intestinal epithelial cells.

Transforming growth factor (TGF)-beta1 has a biphasic effect on rat intestinal epithelial (RIE) cells. By itself, TGF-beta1 functions as a tumor suppressor by inhibiting the growth, migration and invasion of RIE cells. We show in this study that in conjunction with epidermal growth factor (EGF), TGF-beta1 helped to augment migration, invasion and anchorage-independent growth (AIG) compared to that by EGF alone. EGF plus TGF-beta1 induced a dramatic morphological change characteristic of epithelial-mesenchymal transition (EMT). The mechanism for this enhanced effect of TGF-beta1 and EGF on oncogenic properties was explored by analysis of EGF- and TGF-beta1-mediated signaling pathways and complementary DNA arrays. TGF-beta1 augmented EGF-mediated signaling of mitogen-activated protein kinase (MAPK) and AKT by enhancing and prolonging the activation of the former and prolonging the activation of the latter. Inhibition of MAPK, but not phosphoinositide-3 kinase (PI3K), abolished TGF-beta1 plus EGF-induced EMT and downregulation of E-cadherin at mRNA and protein levels. By contrast, cell migration and invasion were sensitive to inhibition of either MAPK or PI3 kinase. TGF-beta1 plus EGF-induced AIG was significantly more resistant to inhibition of PI3K and MAPK compared to that induced by EGF alone. EGF and TGF-beta1 synergistically induced the expression of a series of proteases including matrix metalloproteinase (MMP) 1 (collagenase), MMP3, MMP9, MMP10, MMP14 and cathepsin. Among them, the expression of MMP1, MMP3, MMP9 and MMP10 was MAPK dependent. Inhibition of the MMPs or cathepsin significantly blocked EGF plus TGF-beta1-induced invasion, but had no effect on colony formation. Phospholipase C (PLC) and Cox2 induced by EGF plus TGF-beta1 also played a significant role in invasion, whereas PLC was also important for colony formation. Our study reveals specific signaling functions and induction of genes differentially required for enhanced effect of EGF- and TGF-beta1-induced oncogenic properties, and helps to explain the tumor-promoting effect of TGF-beta1 in human cancer with elevated expression or activation of TGF-beta1 and receptor protein tyrosine kinases.

Ras transformation of RIE-1 cells activates cap-independent translation of ornithine decarboxylase: regulation by the Raf/MEK/ERK and phosphatidylinositol 3-kinase pathways.

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5' untranslated region (5'UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5'UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)-mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G(1). When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity.

Identification of apoptotic genes mediating TGF-beta/Smad3-induced cell death in intestinal epithelial cells using a genomic approach.

Transforming growth factor (TGF)-beta-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-beta inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-beta-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-beta-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-beta/Smad3. Total RNA samples were analyzed using Affymetrix oligonucleotide microarrays. We found that TGF-beta regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-beta activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-beta induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-beta-induced apoptosis in RIE-1/Smad3 cells.