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Objective: Research has highlighted the importance of reward‐based processes in binge eating (BE). However, both increased and decreased activation have been observed in reward related brain regions for BE. Differences may be similar to addiction research, where the reward system is initially hyper‐responsive at early stages of use, but becomes hypo‐responsive with prolonged drug/alcohol use. This study was the first to examine differences in reward system responsivity at early versus chronic BE stages. Method: Using an animal model, Sprague–Dawley female rats were identified as BE prone (BEP) or BE resistant (BER) and randomly assigned to an early or chronic stage group. Neural activation (via Fos protein) was quantified in the nucleus accumbens core (NAC) and shell (NAS). Results: Early stage BEP rats had the highest levels of Fos expression of all of the study groups. By contrast, chronic stage BEP rats exhibited decreased activation in the NAS and NAC that was similar to the activation in chronic stage BER rats. Discussion: Findings are significant in suggesting hyper‐neural activation to reward in the early stages of BE and decreased activation in later stages of BE. Additional studies are needed to elucidate how these differences may impact risk for and maintenance of BE. [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]

Introduction: Muscles add sarcomeres in response to stretch, presumably to maintain optimal sarcomere length. Clinical evidence from patients with cerebral palsy, who have both decreased serial sarcomere number and reduced satellite cells (SCs), suggests a hypothesis that SCs may be involved in sarcomere addition.Methods: A transgenic Pax7-DTA mouse model underwent conditional SC depletion, and their soleii were then stretch-immobilized to assess the capacity for sarcomere addition. Muscle architecture, morphology, and extracellular matrix (ECM) changes were also evaluated.Results: Mice in the SC-reduced group achieved normal serial sarcomere addition in response to stretch. However, muscle fiber cross-sectional area was significantly smaller and was associated with hypertrophic ECM changes, consistent with fibrosis.Conclusions: While a reduced SC population does not hinder serial sarcomere addition, SCs play a role in muscle adaptation to chronic stretch that involves maintenance of both fiber cross-sectional area and ECM structure. Muscle Nerve 55: 384-392, 2017. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a bacterium associated with chronic periodontitis that possesses a family of genes encoding hemagglutinins required for heme acquisition. In this study we generated ΔhagB and ΔhagC mutants in strain W83 and demonstrate that both hagB and hagC are required for adherence to oral epithelial cells. Unexpectedly, a double ΔhagB/ΔhagC mutant had less severe adherence defects than either of the single mutants, but was found to exhibit increased expression of the gingipain-encoding genes rgpA and kgp, suggesting that a ΔhagB/ΔhagC mutant is only viable in populations of cells that exhibit increased expression of genes involved in heme acquisition. Disruption of hagB in the fimbriated strain ATCC33277 demonstrated that HagB is also required for stable attachment of fimbriated bacteria to oral epithelial cells. Mutants of hagC were also found to form defective single and multi-species biofilms that had reduced biomass relative to biofilms formed by the wild-type strain. This study highlights the hitherto unappreciated importance of these genes in oral colonization and biofilm formation. [ABSTRACT FROM AUTHOR]

Objective: To analyze whether a combination of quercetin (Q) and resveratrol (RSV) would induce a white adipose tissue (WAT) browning effect.Methods: Thirty-six rats were fed an obesogenic diet and divided into four groups: control, treated with RSV (15 mg/kg body weight/day; RSV group), treated with Q (30 mg/kg body weight/day; Q group), or treated with both polyphenols (RSV + Q group).Results: After 6 weeks, body and WAT weights were significantly reduced in the RSV + Q group. In perirenal WAT of the control, RSV, and Q groups, white unilocular adipocytes appeared in the majority of cells, while in the RSV + Q group numerous multilocular adipocytes with positive immunostaining for UCP1 were observed. The presence of UCP1 was confirmed by Western blot. This group also revealed increased mRNA levels of Cidea, Hocx9, Bmp4, Slc27a1, Pat2, Atgl, and Atp5d. Interscapular brown adipose tissue weight showed no differences between groups, but the Cidea mRNA level was increased in the RSV group, the Cox-2 mRNA level in the RSV + Q group, and UCP1 protein expression in the RSV and the RSV + Q groups.Conclusions: This study demonstrated that the RSV + Q combination produces a brown-like remodeling effect in perirenal WAT, as well as increased UCP1 protein expression in interscapular brown adipose tissue. [ABSTRACT FROM AUTHOR]

Background: The normal red blood cell (RBC) life span may be significantly reduced when RBCs are stored under blood bank conditions, resulting in a reduced 24-hour survival after transfusion. The damage of stored RBCs is probably multifactorial as stored RBCs share features of both senescence and suicidal RBC death (eryptosis). Since an increased intracellular Ca(2+) concentration ([Ca(2+) ]i ) is one key feature of eryptosis, we here investigated if stored human RBCs had increased [Ca(2+) ]i and the mechanisms behind uptake of such RBCs in a murine model.Study Design and Methods: The intracellular Ca(2+) content of RBCs was determined using the Ca(2+) probe Fluo-3 and flow cytometry. In vivo uptake of Ca(2+) ionophore-treated murine RBCs (Ca(2+) -RBCs) was investigated in recipient mice, using flow cytometry and immunohistochemical analysis.Results: A small fraction of human RBCs accumulated [Ca(2+) ]i during storage for up to 42 days under blood bank conditions. In a murine model, where fresh or Ca(2+) -RBCs were transfused, Ca(2+) -RBCs were mainly trapped by MARCO+ splenic marginal zone macrophages and CD11c+ CD207+ dendritic cells (DCs) within 1 hour after transfusion. In marked contrast, freshly transfused RBCs aging normally in circulation were cleared much slower and preferentially by F4/80+ red pulp macrophages. CD47 on the Ca(2+) -RBCs did not affect their clearance by splenic phagocytic cells.Conclusions: A small fraction of RBCs accumulate [Ca(2+) ]i during storage, and in a murine model such RBCs are recognized by splenic macrophages and DCs in ways similar to what has been reported for nucleated apoptotic cells. [ABSTRACT FROM AUTHOR]

Objective: It is well known that uncoupling protein 1 (UCP1) in brown adipose tissue plays an important role in diet-induced thermogenesis. In this study, whether sarcolipin (SLN), a regulator of sarco/endoplasmic reticulum Ca(2+) -ATPase pump in muscle, is also an important player of diet-induced thermogenesis was investigated, as well as whether loss of SLN could be compensated by increased UCP1 expression and vice versa.Methods: Age- and sex-matched UCP1(-/-) , SLN(-/-) , and double knockout for both UCP1 and SLN mice maintained in C57Bl/6J background were challenged to high-fat diet for 12 weeks and then analyzed for weight gain, alterations in serum metabolites, and changes in thermogenic protein expression.Results: Loss of either SLN or UCP1 alone was sufficient to cause diet-induced obesity. No compensatory upregulation of UCP1 in SLN(-/-) mice or vice versa was found. Paradoxically, loss of both mechanisms failed to exacerbate the obesity phenotype.Conclusions: Data suggest that both SLN- and UCP1-based adaptive thermogenic mechanisms were essential for achieving maximal diet-induced thermogenesis. When both mechanisms were absent, less efficient thermogenic mechanisms were activated to counter energy imbalance. [ABSTRACT FROM AUTHOR]

Background and Purpose: There is emerging evidence suggesting that abnormal transport of amyloid-β (Aβ) across the blood-brain barrier (BBB) is involved in diabetes-associated cognitive decline. We investigated whether PPARγ agonists restore Aβ transport across the BBB and hippocampal plasticity in db/db mice.Experimental Approach: Efflux and influx of Aβ across the BBB were determined by stereotaxic intra-cerebral or i.a. infusion of [(125) I]-Aβ1-40 respectively. Receptor for advanced glycation end products (RAGE) and low-density lipoprotein receptor-related protein 1 (LRP1), which are involved in Aβ influx and efflux, PPARγ and NF-κB p65 at the BBB, as well as hippocampal Aβ, caspase-3, Bax and Bcl-2 were assayed by Western blot, immunohistochemistry and RT-PCR. In vivo, hippocampal LTP was recorded, and Morris water maze and Y-maze tasks were performed.Key Results: Treatment with PPARγ agonists, rosiglitazone (0.8 mg·kg(-1) ) and pioglitazone (9.0 mg·kg(-1) ), for 6 weeks significantly increased Aβ efflux and decreased Aβ influx across the BBB in db/db mice. Concomitantly, they decreased hippocampal Aβ1-40 and Aβ1-42 , suppressed neuronal apoptosis, as indicated by decreased caspase-3 activity and increased ratio of Bcl-2/Bax, and increased hippocampal plasticity, characterized by an enhanced in vivo LTP and better performance in behavioural tests. Furthermore, the PPARγ agonists induced the expression of LRP1 gene by activation of PPARγ and suppressed RAGE gene expression by inactivation of NF-κB signalling at the BBB of db/db mice.Conclusions and Implications: PPARγ agonists modify abnormal Aβ transport across the BBB and this is accompanied by amelioration of β-amyloidosis and an improvement in hippocampal plasticity in diabetic mice. [ABSTRACT FROM AUTHOR]

The homeobox, msh-like 1 (MSX1) protein is essential for cell proliferation and differentiation. Tooth germ development of Msx1 knockout mouse is arrested at the bud stage, impeding an understanding of its role beyond this stage of tooth development. The aims of this study were to investigate the potential role of MSX1 in the regulation of proliferation and differentiation of dental mesenchymal cells in culture, and to preliminarily explore its underlying mechanism of action. Tooth germs were isolated from embryonic day (E)15.5 mice. The mesenchyme was separated and digested into a single-cell suspension, and then cultured in vitro. Isolated dental mesenchymal cells were transfected with MSX1 small interfering RNA, and the effects on cell proliferation, cell cycle distribution, and the expression of bone morphogenetic protein 2 ( Bmp2) and bone morphogenetic protein 4 ( Bmp4) were studied. We also compared the expression levels of alkaline phosphatase ( Alp), type I collagen ( Col1A), osteocalcin ( Ocn), runt-related transcription factor 2 ( Runx2), dentin sialophosphoprotein ( Dspp) and dentin matrix protein 1 ( Dmp1), and mineralized nodule formation, between control and MSX1 siRNA-transfected groups after the induction of odontoblast differentiation. Knockdown of Msx1 expression was associated with decreased cell proliferation, prolonged time in the S phase of the cell cycle, enhanced odontoblast differentiation, and elevated Bmp2 and Bmp4 expression. We conclude that MSX1 may promote proliferation and prevent the differentiation of dental mesenchymal cells by the inhibition of Bmp2 and Bmp4 expression. [ABSTRACT FROM AUTHOR]

The aim of the present study was to clarify an involvement of growth-associated protein-43 (GAP-43) in the regeneration of primary afferent trigeminal ganglion (TG) neurons following inferior alveolar nerve transection (IANX). A larger number of GAP-43 immunoreactive (GAP-43 IR) TG neurons was observed in rats 3 d after IANX compared with sham rats. Growth-associated protein-43 IR TG neurons were also detected for 30 d after IANX, and the number of GAP-43 IR TG neurons was significantly higher in the IANX model until day 30. The relative number of large (>600 μm2) GAP-43 IR TG neurons was significantly lower, whereas the relative number of small (<400 /μm2) GAP-43 IR TG neurons was significantly higher than that at day 0 until 30 d after IANX. To evaluate the functional recovery of damaged IAN, the jaw opening reflex (JOR), elicited by the electrical stimulation of the IAN, was measured before and after IANX. Jaw opening reflex occurrence was gradually increased and the relative threshold of electrical stimulation eliciting JOR was gradually decreased over the 30-d duration of the study. On day 30 after IANX, the JOR occurrence and relative JOR threshold were similar to those in sham rats. The present findings suggest that changes in the expression of GAP-43 in TG neurons after IANX are involved in regeneration and functional recovery of the transected IAN. [ABSTRACT FROM AUTHOR]

Objective and Background Gingival epithelium protects against bacterial infection by producing antimicrobial peptides such as calprotectin. Calprotectin consists of proteins S100 A8 and S100 A9. Although in vitro assay has shown that neutrophils and gingival epithelial cells express calprotectin, the expression of S100 A8 and S100 A9 and colocalization of both S100 proteins in gingival tissue in vivo are not fully understood. The aim of this study was to investigate the distribution of S100 A8 and S100 A9 expression in gingival epithelium of mice in the presence and absence of infection. Materials and Methods A quantitative analysis of S100 A8 and S100 A9 m RNA in junctional epithelium ( JE) and oral gingival epithelium ( OGE) of both germ-free mice and conventional mice was performed using laser microdissection and real-time polymerase chain reaction ( PCR). Confirmation of S100 A8 and S100 A9 m RNA expression in the JE was conducted by fluorescent immunohistochemistry. Results Real-time PCR analysis indicated that S100 A8 and S100 A9 expressions were mainly detected in JE and only slightly or not detected in OGE. Levels of both S100 A8 and S100 A9 m RNA expression in JE of conventional mice were significantly higher than those in JE of germ-free mice. Additionally, fluorescent immunohistochemistry showed that S100 A8 expression was observed in the JE of both conventional and germ-free mice, whereas S100 A9 was expressed in the JE of conventional but not germ-free mice. Conclusion S100 A8 protein is expressed in JE cells of mice in the presence and in the absence of infection with oral bacteria. S100 A9 expression in JE cells in the presence of microflora is significantly increased compared with the absence of microflora, which suggests that S100 A9 expression may be induced by infection of microflora. The production of calprotectin in gingival epithelial cells may be mediated through S100 A9 induction by bacterial infection. [ABSTRACT FROM AUTHOR]

The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts. [ABSTRACT FROM AUTHOR]

The formation of dentin and enamel is initiated by the differentiation of odontogenic precursor cells into odontoblasts and ameloblasts, respectively. This study was performed to identify new molecules involved in the differentiation of odontogenic cells. The genes expressed differentially between the root stage (after the differentiation of odontogenic cells and dental hard-tissue formation) and the cap stage (before the differentiation of odontogenic cells and dental hard-tissue formation) were searched using differential display PCR. For the first time, synaptic vesicle protein ( SV) 2b, an important transmembrane transporter of Ca2+-stimulated vesicle exocytosis, was identified as a differentially expressed molecule. Real-time PCR and western blotting revealed an increase in the transcriptional and translational levels of SV2b during or after the differentiation of odontogenic cells. Immunofluorescence revealed this molecule to be localized in not only fully differentiated odontoblasts but also in pre-odontoblasts before dentin matrix secretion. The expression pattern of the SV2a isoform was similar to that of the SV2b isoform, whereas the SV2c isoform showed a contrasting pattern of expression. After treatment with alendronate, an inhibitor of protein isoprenylation for the transport of secretory vesicles, the expression of SV2a and SV2b decreased, whereas that of SV2c increased. These results suggest that the SV2 isoforms are functional molecules of (pre)odontoblasts which may be involved in vesicle transport. [ABSTRACT FROM AUTHOR]

Keratin gene expression is regarded as a hallmark of epidermal biology. It demarcates the three keratinocyte phenotypes: basal (expressing KRT5 and KRT14), differentiating (expressing KRT1 and KRT10), and activated (wound healing), which is characterized by expression of KRT6, KRT16, and KRT17. Activated keratinocytes are among the first signals of epidermal wound healing. In addition, they are found deregulated in nonhealing chronic wounds. To examine keratins as a potential modality for wound-healing disorders, we evaluated two different keratin dressings, liquid or solid, and assessed their effects of epithelialization and closure using porcine partial-thickness wound-healing model in vivo. We found that both forms of keratin dressings accelerated closure and epithelialization, achieving statistically significant differences on day 5. Evidence suggesting early onset of epithelialization was corroborated further by gene expression analyses revealing induction of KRT6A, KRT16, and KRT17 by day 2 postwounding. The data suggest that keratin dressings may stimulate epithelialization by enhancing the activation of keratinocytes. We conclude that keratin-containing dressings can accelerate wound healing and closure. Further studies are needed to determine the molecular mechanisms of this activation. [ABSTRACT FROM AUTHOR]

The article presents abstracts related to beta cells, stem cells, and islets including the activation of extracellular calcium-sensing receptor (CaR), beta cell function in islet transplantation, and human-islet-derived cell line's characteristics.

Zhang Y, Zhang X, Lu X, Atsawasuwan P, Luan X. Ameloblastin regulates cell attachment and proliferation through RhoA and p27. Eur J Oral Sci 2011; 119 (Suppl. 1): 280-285. © 2011 Eur J Oral Sci The matrix adhesion protein ameloblastin (AMBN) is one of the unique components of the mineralizing matrix of bones and teeth. Here we focused on two types of cells expressing AMBN - mouse dental follicle cells (mDF) and mouse periodontal ligament cells (mPDL) - to decipher AMBN function in developing dental, periodontal, and bone tissues. To test AMBN function, cell culture dishes of mDF and mPDL were exposed to either full-length or C-terminal (amino acids 137-407) recombinant Ambn protein. Alternatively, cells were subjected to transient transfection using an Ambn- small hairpin (sh) RNA vector. Our cell culture studies documented that dishes coated with full-length AMBN promoted the attachment of mPDL and mDF cells as early as 1 h after seeding. In order to identify potential intermediaries that might aid the effect of AMBN on adhesion, RhoA expression levels in AMBN-coated and uncoated control dishes were assessed. These studies indicated that AMBN induced RhoA expression 4 h after seeding, especially in mPDL cells. After 4 h of culture, the cell cycle inhibitor p27 was also up-regulated. In addition, exogenous AMBN and its C-terminal fragment reduced the proliferation of mDF and mPDL. Finally, transient transfection of mDF and mPDL cells with the Ambn-shRNA vector resulted in the down-regulation of p27 in mPDL cells. Together, these data indicate that AMBN affects cell adhesion via RhoA and cell cycle progression through p27. [ABSTRACT FROM AUTHOR]

Oral Diseases (2011) , 489-498 The aim of this study was to investigate how interleukin (IL)-8 (cytokine-induced neutrophil chemoattractant; CINC-1) and monocyte chemotactic protein (MCP)-1/CCL2 contribute to root resorption during orthodontic tooth movement. Forty 6-week-old male Wistar rats were subjected to orthodontic force of 10 or 50 g to induce a mesially tipping movement of the upper first molars for 7 days. We determined the expressions of CINC-1, CXCR2, and MCP-1 proteins in root resorption area using immunohistochemistry. Furthermore, we investigated the effects of compression forces (CF) on IL-8 and MCP-1 production by human periodontal ligament (hPDL) cells. We observed an effect of chemokine treatment on rat odonto/osteoclasts in dentin slices that recapitulated root resorption. The immunoreactivity for CINC-1/CXCR2 and MCP-1 was detected in odontoclasts and PDL fibroblasts by the orthodontic force of 50 g on day 7. CF increased the secretion and the expression of mRNA of IL-8 and MCP-1 from PDL cells in a magnitude-dependent manner. Moreover, CINC-1 and MCP-1 stimulated osteoclastogenesis from rat osteoclast precursor cells. IL-8 (CINC-1) and MCP-1 may therefore facilitate the process of root resorption because of excessive orthodontic force. [ABSTRACT FROM AUTHOR]

Nishio C, Wazen R, Kuroda S, Moffatt P, Nanci A. Disruption of periodontal integrity induces expression of apin by epithelial cell rests of Malassez. J Periodont Res 2010; 45: 709-713. © 2010 John Wiley & Sons A/S It has been suggested that epithelial cell rests of Malassez (ERM) may express enamel matrix proteins and play an important role in periodontal regeneration. Two novel proteins, apin (APIN) and amelotin (AMTN), produced by maturation-stage ameloblasts and junctional epithelium, have recently been identified. The objective of this study was to evaluate whether the ERM express APIN and AMTN under normal conditions and after periodontal challenge. Gingivectomy and orthodontic tooth movement were carried out on the left side of the maxillae of rats. The control group included the untreated contralateral side of these animals and the maxillae of normal, untreated rats. Animals were sacrificed by intracardiac perfusion on days 3 and 5 after the experimental procedures and maxillary molars were decalcified and processed for paraffin embedding. Immunohistochemistry was used to evaluate the expression of various ameloblast products, including APIN, AMTN, ameloblastin (AMBN) and amelogenin (AMEL). At 3 and 5 days after periodontal challenge, ERM were more evident in the periodontal ligament along the root surface and in the root furcations. Immunodetection of APIN, but not of the other three proteins, was observed in the ERM following the disruption of periodontal integrity. No immunolabeling for APIN, AMTN, AMBN and AMEL was detected in the ERM under normal conditions. The expression of APIN at an early time-point following disruption of periodontal integrity suggests that this protein may be part of the cascade of events leading to the activation of ERM during periodontal healing and regeneration. [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 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: Endothelial NO synthase (eNOS) is a dynamic enzyme tightly controlled by co- and post-translational lipid modifications, phosphorylation and regulated by protein-protein interactions. Here we have pharmacologically modulated the activation of eNOS, at different post-translational levels, to assess the role of eNOS-derived NO and of these regulatory mechanisms in intestinal injury associated with splanchnic artery occlusion (SAO) shock.Experimental Approach: SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk for 45 min followed by 30 min of reperfusion. During ischemia, 15 min prior to reperfusion, mice were given geldanamycin, an inhibitor of hsp90 recruitment to eNOS, or LY-294002 an inhibitor of phosphatidylinositol 3-kinase (PI3K), an enzyme that initiates Akt-catalysed phosphorylation of eNOS on Ser1179. After 30 min of reperfusion, samples of ileum were taken for histological examination or for biochemical studies.Key Results: Either LY-294002 or geldanamycin reversed the increased activation of eNOS and Akt observed following SAO shock. These molecular effects were mirrored in vivo by an exacerbation of the intestinal damage. Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity, and with an increased expression of the adhesion proteins: ICAM-I, VCAM, P-selectin and E-selectin.Conclusions and Implications: Overall these results suggest that activation of the Akt pathway in ischemic regions of reperfused ileum is a protective event, triggered in order to protect the intestinal tissue from damage induced by ischaemia/reperfusion through a fine tuning of the endothelial NO pathway. [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& 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. 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: 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]

The article presents abstracts of medical research which include acetyl-CoA carboxylase-1 is a critical regulator of beta cell size and proliferation; co-encapsulation of islets with mesenchymal stem cells improves graft outcome in mice; and exploring the role of glycaemic instability in sudden nocturnal cardiac death in Type 1 diabetes.