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Macrophage activation plays a central role in neoatherosclerosis and in-stent restenosis after percutaneous coronary intervention (PCI). Galectin-3, mainly expressed on macrophages, is an important regulator of inflammation. This study aimed to investigate the effects of berberine (BBR) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage activation and galectin-3 expression and their underlying mechanisms. THP-1-derived macrophages were pretreated with BBR prior to stimulation with ox-LDL. Galectin-3 expression was measured by real-time PCR, Western blotting, and confocal microscopy. Macrophage activation was assessed by lipid accumulation, expression of inflammatory cytokines, and CD11b and CD86. Plasma galectin-3 levels were measured in patients undergoing PCI at baseline and after BBR treatment for 3 months. BBR suppressed ox-LDL-induced upregulation of galectin-3 and macrophage activation. Overexpression of galectin-3 intervened the inhibitory effect of BBR on macrophage activation. BBR activated phospho-AMPK and inhibited phospho-NF-κB p65 nuclear translocation. AMPK inhibition and NF-κB activation abolished the inhibitory effects of BBR on galectin-3 expression and macrophage activation. Combination of BBR and rosuvastatin exerted greater effects than BBR or rosuvastatin alone. However, BBR treatment did not further reduce plasma galectin-3 after PCI in patients receiving standard therapy. In conclusion, BBR alleviates ox-LDL-induced macrophage activation by downregulating galectin-3 via the NF-κB and AMPK signaling pathways. [ABSTRACT FROM AUTHOR]

Background: The role of reactive oxygen species (ROS) in activation of the inflammatory response has been proven in previous study using human gingival fibroblasts (HGFs) to lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) stimulation, but its exact mechanism has not been established. ROS can be generated through increased oxidative phosphorylation. P53 originally identified as a tumor suppressor, has been demonstrated to be associated with energy metabolism. We proposed that LPS-induced inflammatory cytokines release in HGFs is mediated by interaction between P53 and ROS levels.Methods: HGFs were grown in medium with Pg LPS stimulation. Gene expression was performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. HGFs were also processed by immunofluorescence to characterize the localization of P53. ROS was measured using a multimodal microplate reader and immunofluorescence microscopy. Cellular respiration levels were performed with a high-resolution respirometer. Cytokines secretion was confirmed by enzyme-linked immunosorbent assay.Results: LPS-induced P53 activity and localization in mitochondria led to cellular redox imbalance and mitochondrial dysfunction, thus triggered the cellular inflammatory response with increased secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. Furthermore, the cellular redox imbalance and inflammation induced by LPS were reversed by inhibiting P53 activity. P53 expression followed by LPS-induced inflammation was also be restricted by suppressing ROS generation.Conclusions: The present study shows that LPS-induced inflammation in HGFs is partially dependent on P53 modulating ROS and ROS stimulating P53, which suggests that P53 and ROS may form a feedback loop. The identification of this mechanism may provide potential new therapeutic strategies for periodontitis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects.Experimental Approach: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques.Key Results: Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR.Conclusions and Implications: CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent. [ABSTRACT FROM AUTHOR]

Objective: Innate and adaptive immunity may contribute to gland dysfunction in patients with primary Sjögren's syndrome (pSS). The P2X7 receptor (P2X7 R)-NLRP3 inflammasome complex modulates the release of the inflammatory cytokines IL-1β and IL-18. The presence of P2X7 R in salivary glands suggests an interesting scenario for the initiation and amplification of the innate immune response in pSS. Therefore, the aim of this study was to assess the role of the P2X7 R-NLRP3 inflammasome in pSS. Subjects and Methods: Twenty-one consecutive patients with pSS according to the American-European Consensus Group criteria and 15 patients with sicca syndrome (i.e. without Sjögren's syndrome, non-SS) were enrolled in this study, together with six control (CTL) subjects. Expression of the P2X7R-NLRP3 platform and IL-18 was determined by real-time PCR and western blotting in gland specimens and peripheral lymphomonocytes; data were related to patients\x92 clinical, serological and histopathological characteristics. The presence of IL-18 was determined in gland and saliva samples. Results: P2X7 R expression was significantly higher in salivary glands from individuals with pSS than in those from non-SS and CTL subjects. Accordingly, the gene expression levels of the inflammasome components NLRP3, ASC and caspase-1 were significantly higher in pSS gland specimens, and this was paralleled by an increased expression of mature IL-18 in pSS saliva samples. The expression of both the P2X7 R and the inflammasome components was a marker of disease-related glandular involvement, being increased in patients with anti-Ro/SSA positivity and correlated with focus score. Conclusion: The results of this study suggest an involvement of the P2X7 R-inflammasome-caspase-1-IL-18 axis in the development of pSS exocrinopathy. This finding provides the basis for studying the complex mechanisms underlying pSS, as well as for developing novel potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

ACADEMIC EMERGENCY MEDICINE 2012; 19:48-55 © 2012 by the Society for Academic Emergency Medicine Abstract Objectives: Over the past decade, clinicians have become increasingly reliant on computed tomography (CT) for the evaluation of patients with suspected acute appendicitis. To limit the radiation risks and costs of CT, investigators have searched for biomarkers to aid in diagnostic decision-making. We evaluated one such biomarker, calprotectin or S100A8/A9, and determined the diagnostic performance characteristics of a developmental biomarker assay in a multicenter investigation of patients presenting with acute right lower quadrant abdominal pain. Methods: This was a prospective, double-blinded, single-arm, multicenter investigation performed in 13 emergency departments (EDs) from August 2009 to April 2010 of patients presenting with acute right lower quadrant abdominal pain. Plasma samples were tested using the investigational S100A8/A9 assay. The primary outcome of acute appendicitis was determined by histopathology for patients undergoing appendectomy or 2-week telephone follow-up for patients discharged without surgery. The sensitivity, specificity, negative likelihood ratio (LR-), and positive likelihood ratio (LR+) of the biomarker assay were calculated using the prespecified cutoff value of 14 units. A post hoc stability study was performed to investigate the potential effect of time and courier transport on the measured value of the S100A8/A9 assay test results. Results: Of 1,052 enrolled patients, 848 met criteria for analysis. The median age was 24.5 years (interquartile range [IQR] = 16-38 years), 57% were female, and 50% were white. There was a 27.5% prevalence of acute appendicitis. The sensitivity and specificity for the investigational S100A8/A9 assay in diagnosing acute appendicitis were estimated to be 96% (95% confidence interval [CI] = 93% to 98%) and 16% (95% CI = 13% to 19%), respectively. The LR− ratio was 0.24 (95% CI = 0.12 to 0.47), and the LR+ was 1.14 (95% CI = 1.10 to 1.19). The post hoc stability study demonstrated that in the samples that were shipped, the estimated time coefficient was 7.6 × 10−3 ± 2.0 × 10−3 log units/hour, representing an average increase of 43% in the measured value over 48 hours; in the samples that were not shipped, the estimated time coefficient was 2.5 × 10−3 ± 0.4 × 10−3 log units/hour, representing a 13% increase on average in the measured value over 48 hours, which was the maximum delay allowed by the study protocol. Thus, adjusting the cutoff value of 14 units by the magnitude of systematic inflation observed in the stability study at 48 hours would result in a new cutoff value of 20 units and a 'corrected' sensitivity and specificity of 91 and 28%, respectively. Conclusions: In patients presenting with acute right lower quadrant abdominal pain, we found the investigational enzyme-linked immunosorbent assay (ELISA) test for S100A8/A9 to perform with high sensitivity but very limited specificity. We found that shipping effect and delay in analysis resulted in a subsequent rise in test values, thereby increasing the sensitivity and decreasing the specificity of the test. Further investigation with hospital-based laboratory analyzers is the next critical step for determining the ultimate clinical utility of the ELISA test for S100A8/A9 in ED patients presenting with acute right lower quadrant abdominal pain. [ABSTRACT FROM AUTHOR]

Aim: Some patients suffering from aggressive periodontitis (Ag-P) also display neutrophil chemotaxis dysfunction. In this study, we attempted to identify the proteins involved in Ag-P associated with neutrophil chemotaxis dysfunction using proteome analysis. Material and Methods: A two-dimensional fluorescence difference gel electrophoresis system was used to detect differences in protein expression between neutrophils from four patients suffering from Ag-P combined with neutrophil chemotaxis dysfunction and those from four controls. Moreover, the mRNA levels of the proteins identified by the above method were examined in neutrophils from four types of subjects using the real-time polymerase chain reaction: twenty patients suffering from Ag-P with or without the dysfunction, 15 patients with chronic periodontitis, and 15 controls. Results: Four proteins, lactoferrin, caldesmon, heat shock protein 70, and stac, displayed a higher protein expression level in the neutrophils from the patients suffering from Ag-P combined with the neutrophil dysfunction than in those from the control group. The caldesmon mRNA levels in the neutrophils from the patients suffering from Ag-P combined with the neutrophil dysfunction were high compared with those in the neutrophils from the patients suffering from the other two types of periodontitis and those from the control group. Conclusion: Caldesmon may be a marker of Ag-P combined with neutrophil chemotaxis dysfunction. [ABSTRACT FROM AUTHOR]

Background and Purpose: Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention.Experimental Approach: We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins.Key Results: Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases.Conclusion and Implications: While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1. [ABSTRACT FROM AUTHOR]

Background and Purpose: K(v)11.1 channels are involved in regulating cellular excitability in various tissues including brain, heart and smooth muscle. In these tissues, at least two isoforms, K(v)11.1a and K(v)11.1b, with different kinetics, are expressed. K(v)11.1 activators are potential therapeutic agents, but their effects have only been tested on the K(v)11.1a isoform. In this study, the effects of two different K(v)11.1 activators, NS1643 and RPR260243, were characterized on K(v)11.1a and K(v)11.1b channels.Experimental Approach: K(v)11.1a and K(v)11.1b channels were expressed in Xenopus laevis oocytes, and currents were measured using two-electrode voltage clamp. I/V curves and channel kinetics were measured before and after application of 30 µM NS1643 or 10 µM RPR260243.Key Results: NS1643 increased steady-state currents through Kv11.1b several fold more than through K(v)11.1a channels, without affecting EC(50) values. NS1643 increased activation rates and decreased rates of inactivation, recovery from inactivation and deactivation for both channels. Except for activation, where effect of NS1643 was comparable, relative changes were greater for Kv11.1b than for K(v)11.1a. RPR260243 increased steady-state currents only through Kv11.1a channels, but slowed the process of deactivation for both channels primarily by decreasing time constant of slow deactivation. This effect was greater on K(v)11.1b than on K(v)11.1a. Effects of both compounds on heteromeric K(v)11.1a/K(v)11.1b channels were similar to those on K(v)11.1a.Conclusions and Implications: Both NS1643 and RPR260243 displayed differential effects on K(v)11.1a and K(v)11.1b channels, the effects being relatively more pronounced on K(v)11.1b channels. This affirms the importance of testing the effect of K(v)11.1 activators on different channel isoforms. [ABSTRACT FROM AUTHOR]

Background and Purpose: Stress on the endoplasmic reticulum (ER) can trigger rescuer responses such as the unfolded protein response (UPR). However, pharmacological modulators of these ER-regulated stress responses are not well understood. In the present study, we found that amiloride, a potassium-sparing diuretic, has unique properties relating to such stress.Experimental Approach: We treated mouse primary cultured glial cells with amiloride, in the absence and presence of the ER stress-inducing reagents tunicamycin (Tm) or dithiothreitol, and measured UPR and ER stress-induced cell death. IRE1alpha phosphorylation, eIF2alpha phosphorylation, X-box binding protein 1 (XBP1) splicing, glucose regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression by reverse transcription-polymerase chain reaction and Western blotting were used to assess UPR and lactate dehydrogenase activity was determined to measure ER stress-induced cell death.Key Results: Amiloride completely inhibited ER stress-induced activation of IRE1alpha, an ER-localized stress sensor protein, splicing of XBP1, and subsequent expression of GRP78 at the mRNA and protein levels. ER stress induces the phosphorylation of eIF2alpha, leading to the expression of CHOP or an attenuation of translation in cells. Surprisingly, treatment with amiloride alone markedly promoted the phosphorylation but actually inhibited ER stress-induced CHOP expression. Finally, we found that amiloride (200 microM) synergistically enhanced ER stress-induced cell death, which was mediated through caspases. On the other hand, a low dose of amiloride (20 microM) significantly prevented Tm-induced cell death.Conclusions and Implications: These results suggest that amiloride can modulate UPR. They also suggest amiloride to be an important pharmacological agent and provide basic information for understanding and preventing ER stress-related diseases. [ABSTRACT FROM AUTHOR]

Background and Purpose: In mammalian heart, connexin43 (Cx43) represents the predominant connexin in the working myocardium. As the beta-adrenoceptor is involved in many cardiac diseases, we wanted to clarify the pathway by which beta-adrenoceptor stimulation may control Cx43 expression.Experimental Approach: Cultured neonatal rat cardiomyocytes were stimulated with isoprenaline. Cx43 expression as well as activation of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, JUN NH(2)-terminal kinase (JNK) and nuclear translocation of the transcription factors activator protein 1 (AP1) and CRE-binding protein (CREB) were investigated. Additionally, we assessed Cx43 expression and distribution in left ventricular biopsies from patients without any significant heart disease, and from patients with either congestive heart failure [dilated cardiomyopathy (DCM)] or hypertrophic cardiomyopathy (HCM).Key Results: Isoprenaline exposure caused about twofold up-regulation of Cx43 protein with a pEC(50) of 7.92 +/- 0.11, which was inhibited by propranolol, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole) (p38 inhibitor), PD98059 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) (MAPK 1 kinase inhibitor) (Alexis Biochemicals, San Diego, CA, USA) or cyclosporin A. Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44 and JNK, and by translocation of AP1 and CREB to the nucleus. Analysis of Cx43 protein and mRNA in ventricular biopsies revealed that in patients with DCM, Cx43 content was significantly lower, and in patients with HCM, Cx43 content was significantly higher, relative to patients without any cardiomyopathy. More importantly, Cx43 distribution also changed with more Cx43 being localized at the lateral border of the cardiomyocytes.Conclusion and Implication: Beta-adrenoceptor stimulation up-regulated cardiac Cx43 expression via a protein kinase A and MAPK-regulated pathway, possibly involving AP1 and CREB. Cardiomyopathy altered Cx43 expression and distribution. [ABSTRACT FROM AUTHOR]

The free fatty acid receptors FFA2 and FFA3 are recently de-orphanized G protein-coupled receptors that share a group of short-chain free fatty acids as endogenous ligands. The expression of FFA2 and FFA3 by immune cells, in parts of the gastro-intestinal tract and by white adipocytes has suggested their potential as therapeutic targets in conditions including inflammation and obesity. However, although FFA2 and FFA3 display distinct structure-activity relationships for stimulation by short-chain free fatty acids, the overlap between these endogenous agonists and the lack of synthetic small molecule ligands that display selectivity between these two receptors has, until recently, hindered efforts to resolve their individual functions. Recently, chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide has been described as an FFA2 selective ago-allosteric ligand, not only being a direct agonist but also acting as a positive allosteric modulator of the function of short-chain free fatty acids at FFA2. Mutation of a pair of key arginine residues near the top of transmembrane domains V and VII of both FFA2 and FFA3 eliminates the function of short-chain free fatty acids but is without effect on the direct agonist action of chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide at FFA2, confirming the distinct nature of the binding site of the ago-allosteric regulator from the orthosteric binding site for free fatty acids. An understanding of structure-activity relationships for ligands related to chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide is likely to provide greater insight into the mode of action and site of binding of this ligand, but further FFA2 and FFA3 selective ligands, preferably with higher potency/affinity, will be required to fully explore the physiological function of these receptors. [ABSTRACT FROM AUTHOR]

Background: Considering the role of growth factors in periodontal regeneration, the aim of this study was to evaluate the influence of platelet-derived growth factor (PDGF)-BB, insulin-like growth factor (IGF)-1, and transforming growth factor-beta 1 (TGF-beta1), alone or in combination, on the rate of proliferation and adhesion of periodontal ligament (PDL) cells in vitro.Methods: After establishment and characterization of a primary culture of PDL cells, 72 culture dishes were plated with 10(3) cells distributed among four test groups and a control group. Test groups had PDGF-BB, TGF-beta1, IGF-1, or a combination of all three added to the culture medium, whereas the control group received no growth factor. The samples were counted in triplicate 1, 3, 5, and 7 days after seeding. For the adhesion assay, 14 patients provided 30 root fragments distributed among 10 groups: scaling and root planing (SRP), SRP + growth factors, SRP + citric acid plus tetracycline (CA+T), and SRP + (CA+T) + growth factors. The data were evaluated statistically by analysis of variance complemented by Tukey, Dunnett, and Student-Newman-Keuels methods.Results: Maximum rates of proliferation were observed at day 3 for all groups. TGF-beta1 induced a 344.17% +/- 58.80% increased proliferation rate over control (P < 0.05), followed by the combination (277.5% +/- 29.38%), PDGF-BB (238.79% +/- 5.79%), and IGF-1 (233.16% +/- 19.19%). Groups treated by (CA+T) showed increased numbers of cells attached to root fragments, especially SRP + (CA+T) + combination (13.25 +/- 1.79), with significant differences (P < 0.05) from groups treated only by SRP.Conclusion: This combination of growth factors stimulated a mitogenic response and favored the adhesion of PDL cells in vitro, suggesting its possible role in periodontal regeneration. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hepatic stellate cells play an important role in liver fibrosis but little is known about liver myofibroblasts located around the central vein and in the portal area. In this study, intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured to assess the response to endothelin-1 (ET-1), platelet derived growth factor (PDGF) and ATP in rat liver myofibroblasts.Experimental Approach: Rat liver myofibroblasts were compared in 'quiescent' (cultured on Matrigel-coated dishes) and 'activated' (cultured on non-coated plastic dishes) conditions. [Ca(2+)](i) was measured with the fluorescent dye fura-2 and mRNA for ET-1, PDGF and their receptors by RT-PCR.Key Results: ET-1 increased [Ca(2+)](i) in quiescent cells but not in activated cells, whereas PDGF-BB increased [Ca(2+)](i) in activated cells but not in quiescent cells. However, there was no difference between responses to ATP in quiescent or activated cells. ET-1 (in quiescent cells), PDGF-BB (in activated cells) and ATP (in both cells) all induced transient increases in [Ca(2+)](i) in the absence of extracellular Ca(2+) (with EGTA), indicating the involvement of Ca(2+) release from intracellular Ca(2+) stores. The sustained increase in [Ca(2+)](i) in the presence of external Ca(2+) in activated cells (ATP and PDGF) was significantly reduced by nicardipine, a L-type Ca(2+) channel blocker, but not in quiescent cells (ATP and ET-1).Conclusions and Implications: The different pharmacological profiles of [Ca(2+)](i)-response in quiescent and activated myofibroblasts suggest that ET-1 and PDGF contribute differently to myofibroblast activation during the process of liver fibrosis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations.Experimental Approach: Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists.Key Results: When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations.Conclusions and Implications: Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions. [ABSTRACT FROM AUTHOR]

Background: Periodontal ligament (PDL) cells possess osteoblast-like properties and play key roles in periodontal regeneration. Previously, we have reported that ascorbic acid promotes the osteoblastic differentiation of PDL cells by modulating the type I collagen-integrin interaction. However, the signaling pathway activated following collagen-integrin interaction is still unclear. In this study, we examined the involvement of extracellular signal-regulated kinase (ERK)1/2 in the expression of osteoblastic marker genes such as the osteoblast-specific transcriptional factor runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) in PDL cells.Methods: PDL cells were cultured on a conventional or type I collagen-coated dish in the presence or absence of ascorbic acid and examined for ALP activity and osteoblastic marker genes. For detection of ERK1/2, cells were plated on a petri (non-adhesive) dish or type I collagen-coated dish, and Western blot analysis was performed. The effect of the ERK1/2 inhibitor on osteoblastic marker gene expression was examined.Results: Ascorbic acid increased gene expression of Runx2, ALP, and OCN. A combination of ascorbic acid and type I collagen remarkably upregulated Runx2, ALP, and OCN gene expression and ALP activity. Western blot analysis revealed an increased level of ERK1/2 phosphorylation in cells plated on type I collagen. An ERK1/2 inhibitor suppressed ascorbic acid-induced ALP and OCN gene expression, whereas Runx2 was not affected in PDL cells.Conclusion: These results indicate that ERK1/2 is involved in ascorbic acid-induced osteoblastic differentiation during PDL cell attachment to type I collagen. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the clinicopathologic prognostic factors of uterine sarcoma, and determine whether overexpression of p53 and mdm-2 proteins correlate with overall survival and prognostic factors.Methods: Thirty specimens from 43 patients with uterine sarcoma were available for slide review and evaluated by immunohistochemistry at Yonsei and Ajou University Medical Centers for the expression of p53 and mdm-2.Results: Overall survival was found to correlate to histologic type of uterine sarcoma, surgical stage, tumor grade, and mitotic index. Whereas 63% of the sarcomas expressed p53, with a significant correlation to overall survival, 60% expressed mdm-2, with a significant correlation to the mitotic index but not to overall survival.Conclusion: Histologic type, surgical stage, tumor grade, mitotic index, and p53 expression were prognostic factors of the overall survival of patients with uterine sarcoma. [ABSTRACT FROM AUTHOR]

Background& Purpose: Drug-resistant cancer cells frequently display efflux pumps such as P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1) or the transporter ABCG2. These transporters are each capable of mediating the active efflux of numerous anticancer drugs and display relatively distinct substrate preferences. The last, most recently discovered member, ABCG2, plays a major role in resistance in several types of cancer and the precise pharmacology of this multidrug transporter remain unresolved as does the nature of substrate binding.Experimental Approach: Plasma membranes from insect cells expressing ABCG2 were used to characterise binding of [3H]daunomycin to the multidrug transporter. The kinetics of association and dissociation for this substrate and several other compounds were also determined in this experimental system.Key Results: The dissociation constant for [3H]daunomycin binding was 564 +/- 57 nM and a Hill slope of 1.4 suggested cooperative binding. Doxorubicin, prazosin and daunomycin completely displaced the binding of radioligand, while mitoxantrone and Hoechst 33342 produced only a partial displacement. Analysis of the dissociation rates revealed that [3H]daunomycin and doxorubicin bind to multiple sites on the transporter.Conclusions: Both kinetic and equilibrium data support the presence of at least two symmetric drug binding sites on ABCG2, which is distinct from the asymmetry observed for P-gp. The data provide the first molecular details underlying the mechanism by which this transporter is capable of interacting with multiple substrates. [ABSTRACT FROM AUTHOR]

Background: Reparative tissue of extraction sockets was proposed as grafting material in the treatment of periodontal defects. Our hypothesis was that the addition of growth factors to extraction sockets improves the regenerative potential of this tissue when used as a graft. The objective of the present study was to analyze qualitatively and quantitatively the repair of acute Class II furcation defects after they receive this grafting material.Methods: The second and third upper premolars were extracted from four dogs. Platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-I, at concentrations of 6 microg/ml each, were applied to the resulting sockets. After 5 days, 24 acute defects (12 control and 12 test defects) were created in the second, third, and fourth lower premolars. Only the test sites received the graft. The flaps were positioned coronally on both sides and sutured. After 45 days, the specimens were collected, decalcified, and processed histologically in a buccal-lingual plane. The parameters were measured horizontally in the buccal-lingual direction.Results: Repair was histologically and histometrically similar in the two groups. No significant difference was observed between the test and control groups in the parameters connective tissue, new cementum, new bone, and junctional epithelium.Conclusion: The use of this graft did not show beneficial effects on the repair of acute Class II furcation defects in dogs. [ABSTRACT FROM AUTHOR]

1. Vasorelaxation to β2-adrenoceptor stimulation occurs through both endothelium-dependent and endothelium-independent mechanisms, and the former is mediated through Ca2+-independent activation of endothelial-type nitric oxide synthase (NOS-3). Since Ca2+-independent NOS-3 activation may occur through its serine phosphorylation via protein kinase A (PKA) or Akt, we determined the PKA and Akt dependency of β2-adrenergic relaxation of rat aorta. 2. Rat aortic rings were pre-incubated with the PKA inhibitor H-89 (10-7 M), th ephosphatidylinositol 3-kinase (P13K) inhibitor wortmannin (5 x 10-7 M), Akt inhibitor (10-3M), or vehicle, in the absence or presence of the NOS inhibitor N6-nitro-L-arginine methyl ester (L-NAME, 10-4 M). Rings were then contracted with phenylephrine (10-7 M), and concentration relaxation responses determined to the β2-andrenoceptor agonist albuterol. 3. Rings exhibited a concentraion-dependent relaxation to albuterol pEC50 6.9 %±0.2, Emax 88.2 ± 4.0%, L-NAME attenuated Emax to 60.2 ± 3.5% (P<0.001). 4. In the presence of L-NAME, wortmannin or Akt inhibitor did not influence albuterol responses, whereas H-89 reduced Emax further, to 27.5 ± 2.2 % (P<0.0001). 5. In the absence of L-NAME, Emax to albuterol was reduced by H-89, wortmannin or Akt inhibitor, to 56.2 ± 2.2, 56.0 ± 1.6 and 55.4 ± 1.8%, respectively (P<0.001 for each), the combinations H-89 plus wortmannin or H-89 plus Akt inhibitor reduced Emax further still. 6. Western blotting of NOS-3 immunoprecipitates from rat aortas confirmed that albuterol increased serine phosphorylation of NOS-3, and this increase was attenuated by H-89 or Akt inhibitor. 7. Our results indicate that β2-adrenoceptor stimulaion relaxes rat aorta through both NO-dependent and independent mechanisms. The latter is predominantly PKA-mediated, whereas the former occurs through both PKA and P13K/Akt activation. [ABSTRACT FROM AUTHOR]

1. The role of voltage-dependent (IKy) and large conductance Ca2+-activated BKca) K+ currents in the function of the rat aorta was investigated using specific BKca and Ky channel inhibitors in single rat aortic myocytes (RAMs) with patch-clamp technique and in endothelium-denuded aortic rings with isometric tension measurements. 2. The whole-cell K+ currents were recorded in RAMs dialysed with 200 and 444 nM Ca2+ and in perforated-patch configuration. Electrophysiological analysis demonstrasted that IKy appeared at ≥ -40 mV, while BKCa (isolated using 1µM paxilline) were seen positive to -20 mV in all conditions. 3. Voltage-dependent characteristics, but not maximal conductance, of IK, was significantly altered in increased [Ca2+]i. Correolide (1 µM) (a Ky1 channel blocker) did not inhibit the IKv, whereas millimolar concentration of TEA (IC50 = 3.1±0.6 mM, n = 5) and 4-aminopyridine (4-AP), IC50 = 5.9±1.9 mM, n = 7) suppressed IKv. These results and immunocytochemical analysis suggest the Kv2.1 channel to be a molecular correlate for IKv. 4. In nonstimulated aortic rings 1-5 mM TEA and 4-AP (inhibitors of IKv), but not paxilline (1 µM), caused contraction. The frequency of contractile responses to TEA and 4-AP was increased in the presence of 10 mM KCl, which itself did not significantly affect the aortic basal tone. 5. Phenylephrine (15-40 nM) induced sustained tension with superimposed slow oscillatory contractions (termed OWs). OWs were blocked by diltiazem, ryanodine and cyclopiazonic acid, suggesting the involvement of L-type Ca2+ channels and ryanodine-sensitive Ca2+ stores in this process. 6. TEA and 4-AP, but not IbTX, paxilline or correolide, increased the duration and amplitude of OWs, indicating that IK, is involved in the control of oscillatory activity. 7. In conclusion, our findings suggest that the Kv2.1-mediated IKv, and not BKCa, plays an important role in the regulation of the excitability and contractility of rat aorta. [ABSTRACT FROM AUTHOR]

1. Using gene chip technology, we first identified that PGF(2alpha) (FP agonist) and Butaprost (EP(2) agonist) induced about a five-fold upregulation of Nur77 mRNA expression in hFP-HEK 293/EBNA and hEP(2)-HEK293/EBNA cells. Northern Blot analysis revealed that PGF(2alpha)- and Butaprost-induced upregulation of Nur77 expression are dose- and time-dependent. 2. Both PGF(2alpha) and Butaprost upregulated Nur77 gene expression through the protein kinase C (PKC) pathway. These data are the first showing a link between EP(2) receptor stimulation and protein kinase C activation. Calcineurin was found to be involved downstream of the PKC pathway in PGF(2alpha)-induced Nur77 expression, but not in Butaprost-induced Nur77 expression. 3. We also used Nur77 as a marker gene to compare the effects of PGF(2alpha), Butaprost, and Bimatoprost (a prostamide) on Nur77 expression in human primary trabecular meshwork and ciliary smooth muscle (SM) cells, which are target cells for antiglaucoma drugs. The results showed that PGF(2alpha) and Butaprost, but not Bimatoprost, induced upregulation of Nur77 expression in human TM cells. PGF(2alpha), but not Bimatoprost, dramatically induced upregulation of Nur77 mRNA expression in human ciliary SM cells, whereas Butaprost slightly upregulated Nur77 mRNA expression in SM cells. 4. Nur77 promoter deletion analysis indicated that PGF(2alpha), but not Bimatoprost, activated Nur77 promoter-luciferase reporter in hFP-HEK 293/EBNA cells. Butaprost was less efficacious in inducing Nur77 promoter-luciferase reporter activity in hEP(2)-HEK293/EBNA cells relative to PGF(2alpha) in the comparable assay. The data for Nur77 promoter functional analysis were matched to the Northern blot analysis. 5. It appears that PGF(2alpha) and Butaprost activate Nur77 transcription mechanisms through the activation of FP and EP(2) receptor-coupled signaling pathways, whereas Bimatoprost stimulates neither FP nor EP(2) receptors. [ABSTRACT FROM AUTHOR]

1. The 5-HT receptor subtype that mediates bronchocontraction and the involvement of neuronal and non-neuronal acetylcholine was assessed in murine isolated tracheae. 2. Atropine (1-10 nM) caused a rightward shift of the methacholine concentration-effect curves (pA(2)=9.0) but reduced the maximum response to 5-HT, suggesting that 5-HT acts as an indirect agonist. The potency of 5-HT receptor agonists (alpha-methyl-5-HT approximately 5-HT>5-carboxamidotryptamine), together with the competitive antagonism of 5-HT by ketanserin (pA(2)=9.4), suggests the involvement of the 5-HT(2A) receptor. 3. While cholinergic twitch responses to electrical field stimulation were abolished by the fast sodium channel inhibitor tetrodotoxin (300 nM), as well as by combined blockade of N-, P- and Q-type voltage-operated calcium channels by omega-conotoxin GVIA (30 nM) and agatoxin IVA (100 nM), responses to 5-HT were unaffected. Similarly, botulinum toxin A (50 nM) inhibited EFS twitch responses, but not contractions to 5-HT. 4. Choline acetyltransferase immunoreactivity was localised to ganglia and nerve fibres as well as approximately half the epithelial cells in the preparation. Removal of the epithelial layer markedly attenuated the contractile response to 5-HT, but had no effect on contractions to either methacholine or EFS. 5. These findings suggest that 5-HT, acting at 5-HT(2A) receptors on mouse tracheal epithelial cells, stimulates these cells to release acetylcholine, which then causes contraction of airway smooth muscle. This phenomenon should be borne in mind in when interpreting studies of murine models of airway disease. [ABSTRACT FROM AUTHOR]

1 Toluene is a representative example of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this lab and others has shown that toluene modulates the function of N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA) and glycine receptors at concentrations that do not affect non-NMDA receptors. 2 We utilized two-electrode voltage-clamp and whole cell patch-clamp techniques to assess the effects of toluene on neuronal nicotinic acetylcholine receptors expressed in oocytes and cultured hippocampal neurons. Toluene (50 micro M to 10 mM) produced a reversible, concentration-dependent inhibition of acetylcholine-induced current in Xenopus oocytes expressing various nicotinic receptor subtypes. The alpha4beta2 and alpha3beta2 subunit combinations were significantly more sensitive to toluene inhibition than the alpha4beta4, alpha3beta4 and alpha7 receptors. 3 Receptors composed of alpha4 and beta2(V253F) subunits showed alpha4beta4-like toluene sensitivity while those containing alpha4 and beta4(F255V) subunits showed alpha4beta2-like sensitivity. 4 In hippocampal neurons, toluene (50 micro M to 10 mM) dose-dependently inhibited ACh-mediated responses with an IC(50) of 1.1 mM. 5 Taken together, these results suggest that nicotinic receptors, like NMDA receptors, show a subunit-dependent sensitivity to toluene and may represent an important site of action for some of the neurobehavioural effects of toluene. [ABSTRACT FROM AUTHOR]

Background: Short-chain carboxylic acids (SCCA) are metabolic byproducts of anaerobic subgingival bacteria associated with human periodontal disease. We examined the effect of 4 SCCA (butyric, propionic, succinic, and lactic acids) on human polymorphonuclear leukocyte (PMN) apoptosis over the range of concentrations (1 to 30 mM) found in the diseased periodontium.Methods: PMN suspensions were incubated at 37 degrees C with medium alone (control) or one of the 4 SCCA at concentrations of 1, 5, or 30 mM. Aliquots were withdrawn hourly to assess apoptosis and viability by fluorescence microscopy.Results: Relative to untreated controls, PMN incubated for at least 5 hours with 1 mM butyric or propionic acids exhibited significant delays in apoptosis (P<0.05), while those incubated with succinic or lactic acids exhibited no significant differences from controls (P>0.05). At a concentration of 5 mM, propionic, succinic, and lactic acids had little effect on apoptosis (P>0.05), but butyric acid significantly accelerated apoptotic changes (P<0.05). At 30 mM, all SCCA except lactic acid significantly accelerated apoptosis (P<0.05). Incubation with SCCA did not adversely affect cell viability (typically >98%). Lysates from PMN incubated 6 hours with 30 mM butyric or propionic acids contained significantly more caspase-3 activity than lysates from untreated control PMN (P<0.05). Moreover, pretreatment with a specific inhibitor of caspase-3 blocked acceleration of PMN apoptosis by butyric or propionic acids (P<0.05).Conclusion: Low concentrations of butyric or propionic acids delay PMN apoptosis and extend their functional lifespan, while higher concentrations accelerate apoptosis through a mechanism that appears to involve caspase-3. [ABSTRACT FROM AUTHOR]

Background: A challenge in the reconstruction of periodontal structures is the targeted delivery of growth-promoting molecules to the tooth root surface. Polypeptide growth factors such as platelet-derived growth factor (PDGF) stimulate both cementogenesis and osteogenesis. Recent advances in gene therapy offer the advantage of delivering recombinant proteins to tissues for extended periods of time in vivo.Methods: Recombinant adenoviral vectors encoding for the PDGF-A gene were constructed to allow delivery of PDGF transgenes to cells. The recombinant adenoviruses were assembled using the viral backbone of Ad2/CMV/EGFP and replacing GFP (reporter gene encoding green fluorescent protein driven by the cytomegalovirus promoter [CMV] within adenovirus type 2) with the PDGF-A gene. Root lining cells (cloned cementoblasts) were transduced with Ad2/PDGF-A and evaluated for gene expression, DNA synthesis, and cell proliferation. PDGF-inducible genes, c-myc and osteopontin, were also evaluated following gene delivery of Ad2/PDGF-A.Results: The results revealed high level transduction of cementoblasts by gene transfer for 7 days as evidenced by flow cytometry and Northern blotting. Cementoblast DNA synthesis and subsequent proliferation were stimulated by Ad2/PDGF-A at levels equal to or greater than continuous rhPDGF-AA application. Strong message for the PDGF-A gene and protein as evidenced by Northern blotting and immunocytochemistry was noted. Furthermore, the potent induction of c-myc and osteopontin mRNA was found after PDGF gene delivery to cementoblasts.Conclusions: These findings demonstrate that gene delivery of platelet-derived growth factor stimulates cementoblast activity that is sustained above that of rhPDGF-AA application. The use of gene therapy as a mode of growth factor delivery offers a novel approach to periodontal tissue engineering. [ABSTRACT FROM AUTHOR]

Cellular expression of keratins (intermediate filaments) can be demonstrated by immunocytochemistry using unfixed tissue. However, for practical reasons, provision of fresh tissue to the laboratory is often difficult. Recently a fresh tissue transport medium (Patent applied for) has been developed which allows keratin immunocytochemistry to be performed up to 4 days after biopsy. In this study oral mucosal biopsies from 10 patients were hemisected, half placed in the new transport medium at 4 degrees C and the other half immediately frozen in liquid nitrogen. After 4 days frozen sections of both halves of the biopsies were stained by immunocytochemistry using various antikeratin antibodies. The morphology and staining characteristics of the two halves of the biopsies were then assessed. No significant difference could be found in either morphology or preservation of keratin expression in specimens stored in transport medium, as compared to those in liquid nitrogen. This new transport medium may offer considerable advantage for the provision of a histologic and immunocytochemical diagnostic service. [ABSTRACT FROM AUTHOR]