Dentin remineralization using a stimuli-responsive engineered small molecule GSK3 antagonists-functionalized adhesive.

OBJECTIVES: Tideglusib has shown great performance in terms of dentin regenerative properties. This study aims to evaluate bonding ability, of demineralized dentin infiltrated with polymeric nanoparticles (NPs) doped with tideglusib (TG) (TG-NPs). METHODS: Dentin conditioned surfaces were infiltrated with NPs and TG-NPs. Bonded interfaces were created and stored for 24 h and then submitted to mechanical, chemical and thermal challenging. The resin-dentin interface was evaluated through a doubled dye fluorescent technique and a calcium chelator fluorophore under a confocal laser scanning microscopy, and by field emission scanning electron microscopy. RESULTS: Dentin surfaces treated with TG-NPs and load cycled produced higher bond strength than the rest of the groups. Immersion of dentin specimens treated with undoped-NPs in collagenase solution attained the lowest microtensile bond strength (MTBS) values. Both porosity and nanoleakage decreased when dentin was infiltrated with TG-NPs, that revealed strong signals of xylenol orange stain at both hybrid layer and dentinal tubules. The presence of NPs, in general, inducted the presence of mineralized interfaces after mechanical loading and thermocycling. CONCLUSIONS: Nanoparticles doped with tideglusib promoted the highest dentin bonding efficacy among groups, as they facilitated the maximum bond strength values with creation of mineral deposits at the hybrid layer and dentinal walls. Tideglusib enabled scarce porosity, nanoleakage and advanced sealing among dentin groups. SIGNIFICANCE: Doping hydrophilic polymeric NPs with tideglusib, infiltrated in etched dentin represents a reproducible technique to create reparative dentin at the resin-dentin interface, by inducing therapeutic bioactivity.

Hepatic Expression of Phosphorylated Kinase Akt and Glycogen Synthase Kinase-3 Isoforms in Patients with Chronic Hepatitis C.

Some patients with chronic hepatitis C also demonstrate liver steatosis, but the mechanism remains elusive. To analyze the hepatic expression of phosphorylated kinase Akt at Thr 308 and phosphorylated GSK-3 (Glycogen synthase kinase-3) isoforms, GSK3α at Ser 21 and GSK3ß at Ser 9, in chronic hepatitis C patients with normal body weight, glucose, and lipid profiles depending on homeostasis model assessment of insulin resistance (HOMA-IR) levels and histological parameters. The study group consisted of 31 patients with chronic hepatitis C. The hepatic expression of kinase Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) was measured using Western blot assay. Liver steatosis was observed in 41.93% of patients with HCV infection, in those with increased HOMA-IR index (p = 0.02). However, the hepatic expression of Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) was not related to progression of liver steatosis, inflammation, and fibrosis. There was no significant difference in the hepatic expression of kinase Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) in relation to HOMA-IR. Liver steatosis was found to be positively associated with HOMA-IR levels in patients with chronic hepatitis C without metabolic disorders. However, the hepatic expression of Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) did not correspond to progression of liver disease.

Glycogen Synthase Kinase 3ß Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists.

The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3ß (GSK-3ß) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3ß interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3ß redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3ß at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3ß in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3ß. In vitro activity assays demonstrated that GSK-3ß phosphorylation mediated by PKCζ enhanced GSK-3ß activity. We mapped Ser147 of GSK-3ß as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3ß activity, resulting in ß-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3ß basal activity. Thus, we found that PKCζ phosphorylates GSK-3ß at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3ß activity in opposite manners in normal and malignant colon cells.

Clinicopathological significance of wnt/ß-catenin signaling pathway in esophageal squamous cell carcinoma.

BACKGROUND/AIM: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. It has been reported that Wnt signaling pathway plays an important role in Esophageal Cancer progression, metastasis and invasion. However the clinicopathological significance of Wnt2, GSK3ß, and ß-catenin in ESCC has been little reported. In the present study, the aim of this study was to investigate the clinicopathologic and prognosis roles of Wnt2, GSK3ß, and ß-catenin in ESCC tissue. METHODS: 265 ESCC samples were analyzed by immunohistochemistry using Wnt2, GSK3ß, and ß-catenin antibodies. Then, correlation of Wnt2, GSK3ß, and ß-catenin expression with clinicopathological features and prognosis of ESCC patients was statistically analyzed. RESULTS: Cytoplasmic Wnt2 overexpression was detected in 55.5% (147 of 265) ESCCs, which was significantly correlated with the degree of differentiation (P=0.031). Cytoplasmic GSK3ß overexpression was detected in 7.2% (19 of 265) ESCCs, and aberrant ß-catenin expression was identified in 54.3% (144 of 265) of ESCCs. The positive rate of Wnt2 significantly increased with the malignant degree of Kazak ESCC patients. The aberrant ß-catenin expression in GSK3ß-negative ESCC was significantly associated with the ethnic, tumor size, tumor location, degree of differentiation, AJCC stage, lymph node status. Furthermore, the expression of ß-catenin implicated the ethnic difference (P=0.019). In Kaplan-Meier curve analysis, no significant correlation was observed between the expression of Wnt2, GSK3ß, ß-catenin and the poor prognosis of ESCCs. CONCLUSION: The aberrant ß-catenin expression could be an adverse underlying factor in carcinogenesis and progression of ESCC. There was a different statistical signification for ß-catenin in Kazakhs to compare with Hans.

Differential expression of GSK3ß and pS9GSK3ß in normal human tissues: can pS9GSK3ß be an epithelial marker?

Glycogen synthase kinase 3ß (GSK3ß) and phosphorylated GSK3ß at Ser9 (pS9GSK3ß) are crucial in cellular proliferation and metabolism. GSK3ß and pS9GSK3ß are deregulated in many diseases including tumors. Data on altered expression of GSK3ß and pS9GSK3ß are mainly limited to tumor tissues, thus the expression of GSK3ß and pS9GSK3ß in normal human tissue has been largely unknown. Thus, we examined the immunohistochemical localization of GSK3ß and pS9GSK3ß in human fetal and adult tissues, and also compared the expression pattern of GSK3ß and pS9GSK3ß with that of the CK7 and CK20. We found GSK3ß expression in neurons of brain, myenteric plexus in gastrointestinal tract, squamous epithelium of skin, and mammary gland. The expression of pS9GSK3ß was restricted to the epithelial cells of breast and pancreaticobiliary duct, distal nephron of kidney, gastrointestinal tract, fallopian tube, epididymis, secretory cell of prostatic gland, and umbrella cell of urinary tract. The staining pattern of pS9GSK3ß and CK7 was overlapped in most organs except for gastrointestinal tract where CK7 was negative and CK20 was positive. Our results show that the expression of GSK3ß may be associated with differentiation of ectodermal derived tissues and pS9GSK3ß with that of epithelial cells of endodermal derived tissues in human. In addition, the expression of pS9GSK3ß in the selective epithelial cells may indicate its association with secretory or barrier function of specific cells and may serve as another immunohistochemical marker for epithelial cells.

Comparison of the neuropsychological mechanisms of 2,6-diisopropylphenol and N-methyl-D-aspartate receptor antagonist against electroconvulsive therapy-induced learning and memory impairment in depressed rats.

The present study aimed to examine the neurophysiological mechanisms of the 2,6-diisopropylphenol and N-methyl-D-aspartate (NMDA) receptor antagonist against learning and memory impairment, induced by electroconvulsive therapy (ECT). A total of 48 adult depressed rats without olfactory bulbs were randomly divided into six experimental groups: i) saline; ii) 10 mg/kg MK­801; iii) 10 mg/kg MK­801 and a course of ECT; iv) 200 mg/kg 2,6­diisopropylphenol; v) 200 mg/kg 2,6­diisopropylphenol and a course of ECT; and vi) saline and a course of ECT. The learning and memory abilities of the rats were assessed using a Morris water maze 1 day after a course of ECT. The hippocampus was removed 1 day after assessment using the Morris water maze assessment. The content of glutamate in the hippocampus was detected using high­performance liquid chromatography. The expression levels of p­AT8Ser202 and GSK­3ß1H8 in the hippocampus were determined using immunohistochemical staining and western blot analysis. The results demonstrated that the 2,6­diisopropylphenol NMDA receptor antagonist, MK­801 and ECT induced learning and memory impairment in the depressed rats. The glutamate content was significantly upregulated by ECT, reduced by 2,6­diisopropylphenol, and was unaffected by the NMDA receptor antagonist in the hippocampus of the depressed rats. Tau protein hyperphosphorylation in the hippocampus was upregulated by ECT, but was reduced by 2,6­diisopropylphenol and the MK­801 NMDA receptor antagonist. It was also demonstrated that 2,6­diisopropylphenol prevented learning and memory impairment and reduced the hyperphosphorylation of the Tau protein, which was induced by eECT. GSK­3ß was found to be the key protein involved in this signaling pathway. The ECT reduced the learning and memory impairment, caused by hyperphosphorylation of the Tau protein, in the depressed rats by upregulating the glutamate content.

A novel role for GSK3 in the regulation of the processes of human labour.

Preterm birth remains the largest single cause of neonatal death and morbidity. Infection and/or inflammation are strongly associated with preterm delivery. Glycogen synthase kinase 3 (GSK3) is known to be a crucial mediator of inflammation homeostasis. The aims of this study were to determine the effect of spontaneous human labour in foetal membranes and myometrium on GSK3α/ß expression, and the effect of inhibition of GSK3α/ß on pro-labour mediators in foetal membranes and myometrium stimulated with Toll-like receptor (TLR) ligands and pro-inflammatory cytokines. Term and preterm labour in foetal membranes was associated with significantly decreased serine phosphorylated GSK3α and ß expression, and thus increased GSK3 activity. There was no effect of term labour on serine phosphorylated GSK3ß expression in myometrium. The specific GSK3α/ß inhibitor CHIR99021 significantly decreased lipopolysaccharide (ligand to TLR4)-stimulated pro-inflammatory cytokine gene expression and release; COX2 gene expression and prostaglandin release; and MMP9 gene expression and pro MMP9 release in foetal membranes and/or myometrium. CHIR99021 also decreased FSL1 (TLR2 ligand) and flagellin (TLR5 ligand)-induced pro-inflammatory cytokine gene expression and release and COX2 mRNA expression and prostaglandin release. GSK3ß siRNA knockdown in primary myometrial cells was associated with a significant decrease in IL1ß and TNFα-induced pro-inflammatory cytokine and prostaglandin release. In conclusion, GSK3α/ß activity is increased in foetal membranes after term and preterm labour. Pharmacological blockade of the kinase GSK3 markedly reduced pro-inflammatory and pro-labour mediators in human foetal membranes and myometrium, providing a possible therapeutics for the management of preterm labour.

Effect of atorvastatin on wound healing in rats.

Skin-wound healing is a complex and dynamic biological process involving inflammation, proliferation, and remodeling. Recent studies have shown that statins are new therapeutical options because of their actions, such as anti-inflammatory and antioxidant activity, on vasodilation, endothelial dysfunction and neoangiogenesis, which are independent of their lipid-lowering action. Our aim was to investigate the effect of atorvastatin on tissue repair after acute injury in healthy animals. Rats were divided into four groups: placebo-treated (P), topical atorvastatin-treated (AT), oral atorvastatin-treated (AO), topical and oral atorvastatin-treated (ATO). Under anesthesia, rats were wounded with an 8-mm punch in the dorsal region. Lesions were photographed on Days 0, 1, 3, 7, 10, 12, and 14 post-injury and samples taken on Days 1, 3, 7, and 14 for protein-expression analysis of insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase (GSK)-3, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), extracellular signal-regulated kinase (ERK), interleukin (IL)-10, IL-1ß, IL-6, and tumor necrosis factor (TNF)-α. Upon macroscopic examination, we observed significant reductions of lesion areas in groups AT, AO, and ATO compared to the P group. Additionally, AT and AO groups showed increased expression of IRS-1, PI3K, Akt, GSK-3, and IL-10 on Days 1 and 3 when compared with the P group. All atorvastatin-treated groups showed higher expression of IRS-1, PI3K, Akt, GSK-3, IL-10, eNOS, VEGF, and ERK on Day 7. On Days 1, 3, and 7, all atorvastatin-treated groups showed lower expression of IL-6 and TNF-α when compared with the P group. We conclude that atorvastatin accelerated tissue repair of acute lesions in rats and modulated expressions of proteins and cytokines associated with cell-growth pathways.

Unremitting cell proliferation in the secretory phase of eutopic endometriosis: involvement of pAkt and pGSK3ß.

OBJECTIVE: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. DESIGN: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated glycogen synthase kinase 3 beta (pGSK3ß), throughout the menstrual cycle. RESULTS: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3ß was identical in all samples and cycle phases, whereas pAkt and pGSK3ß, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. CONCLUSION: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways.

Sensitization of cancer cells through reduction of total Akt and downregulation of salinomycin-induced pAkt, pGSk3ß, pTSC2, and p4EBP1 by cotreatment with MK-2206.

MK-2206 is an inhibitor of Akt activation. It has been investigated as an anticancer drug in clinical trials assessing the potential of pAkt targeting therapy. The purpose of this study was to identify conditions that increase the sensitivity of cancer cells to MK-2206. We found that the treatment of cancer cells with a high concentration of salinomycin (Sal) reduced total Akt protein levels but increased activated Akt levels. When cancer cells were cotreated with MK-2206 and Sal, both pAkt and total Akt levels were reduced. Using microscopic observation, an assessment of cleaved PARP, FACS analysis of pre-G1 region, and Hoechst staining, we found that Sal increased apoptosis of MK-2206-treated cancer cells. These results suggest that cotreatment with MK-2206 and Sal sensitizes cancer cells via reduction of both pAkt and total Akt. Furthermore, cotreatment of cancer cells with Sal and MK-2206 reduced pp70S6K, pmTOR, and pPDK1 levels. In addition, Sal-induced activation of GSK3ß, TSC2, and 4EBP1 was abolished by MK-2206 cotreatment. These results suggest that cotreatment using MK-2206 and Sal could be used as a therapeutic method to sensitize cancer cells through targeting of the PI3K/Akt/mTOR pathway. Our findings may contribute to the development of MK-2206-based sensitization therapies for cancer patients.

Glycogen synthase kinase-3ß is associated with the prognosis of hepatocellular carcinoma and may mediate the influence of type 2 diabetes mellitus on hepatocellular carcinoma.

BACKGROUND: Although many studies have shown glycogen synthase kinase-3ß (GSK-3ß) was associated with type 2 diabetes mellitus (T2DM) and implicated with a wide range of cancers, the role of GSK-3ß in hepatocellular carcinoma(HCC) and the correlation among GSK-3ß, T2DM and HCC remains unclear. Our objectives were to identify the effect of p-Ser9-GSK-3ß on the prognosis of patients with HCC and to learn more about the interaction among T2DM, GSK-3ß and the prognosis of HCC. METHODS: Firstly we used reverse transcriptase-PCR(RT-PCR) and western blotting to determine the expression levels of GSK-3ß and p-Ser9-GSK-3ß in human HCC samples. We then used immunohistochemical staining to evaluate the expression pattern of p-Ser9-GSK-3ß in 178 patients with HCC after curative partial hepatectomy. Finally we statistically analyzed the association of p-Ser9-GSK-3ß and T2DM with the prognosis of patients with HCC. RESULTS: P-Ser9-GSK-3ß was over-expressed in tumor tissues compared with their normal counterparts. Correlation and regression analysis indicated that the over-expression of p-Ser9-GSK-3ß was significantly associated with T2DM, and the correlation coefficient was 0.259 (P = 0.001). Multivariate analysis showed that the over-expression of p-Ser9-GSK-3ß(P<0.001) and T2DM(P = 0.008) were independently associated with poor prognosis of HCC, respectively. Further analysis demonstrated that these two variables are closely related with each other. CONCLUSION: The over-expression of p-Ser9-GSK-3ß and T2DM are strongly correlated with worse surgical outcome of HCC. P-Ser9-GSK-3ß may play a significant role in mediating the influence of T2DM on the prognosis of HCC.

Pioglitazone ameliorates intracerebral insulin resistance and tau-protein hyperphosphorylation in rats with type 2 diabetes.

OBJECTIVE: To investigate intracerebral insulin resistance and its relationship with tau-protein hyperphosphorylation. METHODS: A rat model of type 2 diabetes (T2D) was established with streptozotocin (STZ). Diabetic rats received intragastric administration of pioglitazone (PIO group) or normal saline (T2D group) for 4 weeks. As a control, non-diabetic rats received intragastric normal saline (CTL group). The insulin concentrations in cerebrospinal fluid (CSF) and blood were determined with radioimmunoassay, and blood glucose concentration was determined using a glucose oxidation technique. Total and phosphorylated levels of protein kinase B (AKT), glycogen synthase kinase-3ß (GSK-3ß) and tau-protein in the hippocampus were analyzed using western blotting. RESULTS: The plasma insulin level in the T2D group was higher, and the CSF insulin level in the T2D group lower than in the CTL group. Hippocampal phosphorylated AKT and phosphorylated GSK-3ß levels were significantly lower in the T2D group than in the CTL group. Hippocampal tau-protein in the T2D group was hyperphosphorylated at Ser199 and Ser396. Plasma insulin levels in the PIO group were lower than in the T2D group, with no differences in CSF insulin levels. Phosphorylated AKT and phosphorylated GSK-3ß levels in the PIO group were significantly higher than in the T2D group Hippocampal phosphorylated tau-protein (Ser199/Ser396) was lower in the PIO group than in the T2D group. CONCLUSION: Hyperphosphorylation of tau-protein in pioglitazone-treated rats with T2D was improved. Rats with T2D have both cerebral insulin resistance and cerebral hypoinsulinism. Pioglitazone can ameliorate intracerebral insulin resistance and decrease tau-protein hyperphosphorylation, but cannot increase intracerebral insulin levels.

Glycogen synthase kinase 3 in chronic rhinosinusitis: two faces of a single enzyme in one disease.

BACKGROUND: The origin and pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) remain unclear. Glycogen synthase kinase 3 (GSK-3) is a unique multitasking kinase involved in the regulation of inflammation and apoptosis and is an important messenger in the downstream signaling of interleukin 6. OBJECTIVE: To analyze the possible role of GSK-3 in the pathogenesis of CRSwNP. METHODS: We examined tissue samples of nasal polyps and the inferior turbinate of patients with CRSwNP and the inferior turbinate of individuals without chronic sinusitis (healthy mucosa). Expression levels of GSK-3 and its inactivated form phosphorylated GSK-3 (pGSK-3) were analyzed using DNA microarray, protein array, Western hybridization, and immunohistochemical analysis. RESULTS: We found increased expression of GSK-3 in both the nasal polyps and the inferior turbinate of patients with CRSwNP compared with those with healthy mucosa (P < .01). We did not observe a difference between nasal polyps and the inferior turbinate of patients with CRSwNP, but a highly significant increase in the phosphorylation rate of GSK-3 was detected in the tissue of nasal polyps compared with the turbinates of patients with CRSwNP (P < .01). CONCLUSION: GSK-3 may play a crucial role in the inflammatory process in CRSwNP. Nasal polyps originate mainly in the mucosa of the middle meatus of the nose and rarely occur in the region of the inferior turbinate. The inhibition of GSK-3 by phosphorylation in nasal polyps, in contrast to the inferior turbinate, is a possible explanation for the different behavior of the mucosa of the middle meatus and the inferior turbinate.

Haloperidol and olanzapine mediate metabolic abnormalities through different molecular pathways.

The pathogenesis of antipsychotic-induced disturbances of glucose homeostasis is still unclear. Increased visceral adiposity has been suggested to be a possible mediating mechanism. The aim of this study was to investigate, in an animal model, the differential effects of olanzapine and haloperidol on visceral fat deposition (using magnetic resonance imaging(MRI)) and on critical nodes of the insulin signaling pathway (liver-protein levels of IRS2 (insulin receptor substrate 2), GSK3α (glycogen synthase kinase-3α), GSK3ß, GSK3α-Ser21, GSK3ß-Ser9). To this end, we studied male Sprague-Dawley rats treated with vehicle (n=8), haloperidol (2 mg kg(-1) per day, n=8), or olanzapine (10 mg kg(-1)per day, n=8), using osmotic minipumps, for 8 weeks. The haloperidol group showed a higher percentage of visceral fat than both the olanzapine group and the vehicle group, whereas there was no difference between the olanzapine and the vehicle group. In terms of insulin signaling pathway, the olanzapine group showed significantly reduced IRS2 levels, reduced phosphorylation of GSK3α and increased phosphorylation of GSK3ß, whereas there was no difference between the haloperidol and the vehicle group. Our data suggest that different molecular pathways mediate the disturbances of glucose homeostasis induced by haloperidol and olanzapine with a direct effect of olanzapine on the insulin molecular pathway, possibly partly explaining the stronger propensity of olanzapine for adverse effects on glucose regulation when compared with haloperidol in clinical settings.

Reduced WIF-1 expression stimulates skin hyperpigmentation in patients with melasma.

The expression of Wnt inhibitory factor-1 (WIF-1) gene, which was detected by a microarray analysis of hyperpigmented and normally pigmented skin sets of melasma patients, was significantly reduced in the hyperpigmented skin from melasma patients, but not in healthy controls, regardless of UV irradiation. Wnt signals regulate skin pigmentation; however, WIF-1 is expressed in cultured skin keratinocytes and fibroblasts, but not in melanocytes. Therefore, we examined whether WIF-1 knockdown in neighboring keratinocytes and fibroblasts plays a role in melasma. Additionally, the effect of WIF-1 overexpression on the amelioration of hyperpigmentation was examined. WIF-1 knockdown, either in fibroblasts or in keratinocytes, significantly stimulated tyrosinase expression and melanosome transfer, whereas melanocytes with WIF-1 overexpression significantly reduced those parameters. The WIF-1 knockdown decreased glycogen synthase kinase-3ß (GSK-3ß), ß-catenin, and NFATc2 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2) phosphorylation and increased microphthalmia-associated transcription factor (MITF) expression as in melanocytes with Wnt-1 overexpression, whereas the WIF-1 overexpression reversed the results. Expression of Wnts, both canonical and noncanonical, was increased in the hyperpigmented skin of melasma patients. Collectively, WIF-1 downregulation, which may occur in epidermal keratinocytes and in dermal fibroblasts, is involved in melasma development because of the stimulation of melanogenesis and melanosome transfer through upregulation of the canonical and the noncanonical Wnt signaling pathway.

GSK3 and ß-catenin determines functional expression of sodium channels at the axon initial segment.

Neuronal action potentials are generated through voltage-gated sodium channels, which are tethered by ankyrinG at the membrane of the axon initial segment (AIS). Despite the importance of the AIS in the control of neuronal excitability, the cellular and molecular mechanisms regulating sodium channel expression at the AIS remain elusive. Our results show that GSK3α/ß and ß-catenin phosphorylated by GSK3 (S33/37/T41) are localized at the AIS and are new components of this essential neuronal domain. Pharmacological inhibition of GSK3 or ß-catenin knockdown with shRNAs decreased the levels of phosphorylated-ß-catenin, ankyrinG, and voltage-gated sodium channels at the AIS, both "in vitro" and "in vivo", therefore diminishing neuronal excitability as evaluated via sodium current amplitude and action potential number. Thus, our results suggest a mechanism for the modulation of neuronal excitability through the control of sodium channel density by GSK3 and ß-catenin at the AIS.

[Expression of RAGE in Helicobacter pylori infested gastric biopsies]. / Lesiones gástricas en pacientes infectados con Helicobacter pylori: expresión de RAGE (receptor de productos de glicosilización avanzada) y otros inmunomarcadores.

BACKGROUND: Inflammation is a common phenomenon present in gastric mucosa of patients infected with H. pylori. Activation of the RAGE/multiligand axis is thought to be a relevant factor in cancer-mediated inflammation. RAGE is a membrane receptor, belonging to the immunoglobulin family, and the over-expression of RAGE has been associated with increased invasiveness and metastasis generation in different types of cancer, including gastric cancer. Furthermore recent experiences show that the use of its soluble form (sRAGE) or silencing of the gene coding for this receptor could provide therapeutic benefits in cancer. AIM: To evaluate the immunohistochemical expression of RAGE, MUC-1, ß-Catenin free and phosphorylated, Cyclin-D1 and GSK3 in gastric biopsy specimens infected with H. pylori. MATERIAL AND METHODS: Immunohistochemical analysis was carried out in gastric biopsies from 138 patients: 55 with inflammatory injury (no atrophic gastritis), 42 with pre-cancerous conditions (atrophy or intestinal metaplasia) and 41 with dysplastic lesions or in situ adenocarcinoma. RESULTS: There was a high rate of positive RAGE expression in the three groups of biopsies. Biopsies with dysplasia or in situ carcinoma had a significantly higher percentage of RAGE expression than the other groups of biopsies. CONCLUSIONS: The increased RAGE expression reported in both dysplasia and incipient cancer support the role of the multiligand/RAGE axis in gastric carcinogenesis.

Noxious stimulation attenuates ketamine-induced neuroapoptosis in the developing rat brain.

BACKGROUND: Ketamine induces neuroapoptosis in neonatal rodents. However, these experimental paradigms were performed without concurrent noxious stimulation, a condition that does not reflect the interaction of anesthesia and surgical stimulation. Noxious stimulation with and without concurrent analgesic drugs has been shown to have divergent patterns of neuronal activation and cell death. We hypothesized that concurrent noxious stimulation would attenuate ketamine-induced caspase-3 activation. METHODS: Postnatal day 7 Sprague-Dawley rat pups were randomized to a 6-h exposure to ketamine with and without peripheral noxious stimulation by intraplantar injection of complete Freund's adjuvant. A cohort of naïve rat pups with and without complete Freund's adjuvant injections served as control subjects. Neuroapoptosis was measured by cleaved caspase-3 expression and terminal deoxynucleotidyl-transferase mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining. In order to determine if concurrent noxious simulation altered the expression of cell survival and cell cycle proteins, levels of protein kinase B and glycogen synthase kinase-3ß and cyclin D1 were measured. RESULTS: Ketamine induced a significant increase in cleaved caspase-3 expression and terminal deoxynucleotidyl-transferase mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining with increases in cyclin D1 levels. Concurrent noxious stimulation with ketamine attenuated caspase-3 activation and maintained cyclin D1 levels. Phosphorylation of protein kinase B and glycogen synthase kinase-3ß was not definitively altered under these conditions. CONCLUSION: The administration of ketamine with concurrent noxious stimulation results in the attenuation of the neuroapoptotic response. These findings suggest that concurrent surgery and procedural pain attenuates ketamine-induced neuroapoptosis.

Effects of herpes simplex virus type 1 infection on immune functions of human neutrophils.

BACKGROUND AND OBJECTIVE: Herpesviruses may play roles in the development of periodontal diseases. This study analyzed the effects of herpes simplex virus type 1 (HSV-1) infection on neutrophil function. The effects of lipopolysaccharide (LPS) from the periodontal pathogen, Porphyromonas gingivalis, during HSV-1 infection were also determined. MATERIAL AND METHODS: Purified HSV-1 was pretreated with buffer containing no serum, with HSV-1 immunoglobulin G (IgG)-positive serum (HSV-1 antiserum) or with control serum. Neutrophils were mock-infected or infected with the pretreated HSV-1. Viral binding and phagosome formation were detected using immunostaining. Intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescin diacetate and fluorometry. Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) were detected using enzyme immunoassays. Release of matrix metalloproteinase-9 (MMP-9) was examined using gelatin zymography. Phosphorylation of Akt/glycogen synthase kinase-3 (GSK-3) was determined using western blotting. RESULTS: HSV-1 bound directly to neutrophils and enhanced the release of MMP-9. HSV-1 immune complexes, formed in the HSV-1 antiserum, bound neutrophils and induced the formation of early phagosome more effectively than did HSV-1 alone. The relative levels of ROS and phosphorylation of Akt/GSK-3 were increased significantly in neutrophils after infection with HSV-1 immune complexes. Infection with HSV-1 and HSV-1 immune complexes also stimulated the production of inflammatory mediators, LTB(4) and IL-8. Moreover, LPS enhanced the HSV-1-stimulatory production of IL-8. CONCLUSION: This study demonstrated differences in neutrophils infected with HSV-1 alone or with HSV-1 immune complexes, suggesting that opsonization of HSV-1 might enhance its effects on neutrophils. The in vitro findings suggest that HSV-1 infection may induce the inflammatory response and affect periodontal health.

Aberrant expression of ß-catenin and its association with ΔNp63, Notch-1, and clinicopathological factors in oral squamous cell carcinoma.

The present study focuses on the correlation between the expression pattern of ß-catenin (component of Wnt signaling), ΔNp63 (proliferation marker), and Notch 1 (transmembrane receptor) in oral squamous cell carcinoma. The study also aims to investigate the interaction between ß-catenin and ΔNp63 in oral cancer. Furthermore, we also analyzed the prognostic significance of ß-catenin, ΔNp63, and Notch 1 in oral squamous cell carcinoma. Immunohistochemical analysis of ß-catenin, ΔNp63, and Notch 1 were done in 62 cases of oral squamous cell carcinoma. Co-immunoprecipitation analysis was done to study the possible interaction between ß-catenin and ΔNp63 in oral cancer. Kaplan-Meier method was used to estimate overall and disease-free survival, and the Log-rank test was used to compare the resulting curves. Statistically significant positive correlation was found between the localization of ß-catenin and the expression of ΔNp63 (p = 0.001**, r (s) = 0.427), whereas, no significant association was found between the expression pattern of ß-catenin and Notch 1. Interestingly, interaction between ß-catenin and ΔNp63 was observed in oral carcinoma. Moreover, ß-catenin and ΔNp63 may be related to worst survival in oral carcinoma. Statistically significant positive association between localization of ß-catenin and expression of ΔNp63 suggests that they might have dependent roles in maintaining the proliferation of oral carcinoma cells. In addition, the downregulated expression of Notch 1 was related to invasion and differentiation status of oral carcinoma cells. Furthermore, ß-catenin and ΔNp63 may be used as independent prognostic markers of oral carcinoma. On the other hand, interaction of ß-catenin with ΔNp63 may be a key event in maintaining the proliferation of oral carcinoma cells. The present study indicates that ß-catenin and ΔNp63 may be used as independent prognostic markers of oral carcinoma and the interaction of ß-catenin with ΔNp63 may be a crucial event in regulating proliferation and differentiation of oral carcinoma cells, which may be used as a target for therapeutic implications.