Long-term sertraline treatment increases expression and decreases phosphorylation of glycogen synthase kinase-3B in platelets of patients with late-life major depression

Background: Abnormal regulation of glycogen synthase kinase 3-beta (GSK3B) activity has been implicated in the pathophysiology of mood disorders. Many pharmacological agents, including antidepressants, can modulate GSK3B. The aim of the present study was to investigate the effect of short-and long-term sertraline treatment on the expression and phosphorylation of GSK3B in platelets of patients with late-life major depression. Methods: Thirty-nine unmedicated elderly adults with major depressive disorder (MOD) were initially included in this study. The comparison group comprised 18 age-matched, healthy individuals. The expression of total and Ser-9 phosphorylated GSK3B (pGSK3B) was determined by Enzyme Immunometric Assay (EIA) in platelets of patients and controls at baseline, and after 3 and 12 months of sertraline treatments for patients only. During this period, patients were continuously treated with therapeutic doses of sertraline. GSK3B activity was indirectly estimated by calculating the proportion of inactive (phosphorylated) forms (pGSK3B) in relation to the total expression of the enzyme (i.e.. GSK3B ratio). Results: Depressed patients had significantly higher levels of pGSK3B as compared to controls (p < 0.001). Within the MDD group, after 3 months of sertraline treatment no significant changes were observed in GSK3B expression and phosphorylation state, as compared to baseline levels. However, after 12 months of treatment we found a significant increase in the expression of total GSK3B (p = 0.05), in the absence of any significant changes in pGSK3B (p = 0.12), leading to a significant reduction in GSK3B ratio (p = 0.001). Conclusions: Our findings indicate that GSK3B expression was upregulated by the continuous treatment with sertraline, along with an increment in the proportion of active forms of the enzyme. This is compatible with an increase in overall GSK3B activity, which may have been induced by the long-term treatment of late-life depression with sertraline. (C) 2012 Elsevier Ltd. All rights reserved.

The inactive form of glycogen synthase kinase-3 beta is associated with the development of carcinomas in galectin-3 wild-type mice, but not in galectin-3-deficient mice

Galectin-3 has been implicated in the tumor development via its mediation of the Wnt signaling pathway. Likewise, glycogen synthase kinase-3beta (GSK3 beta) also plays a role in the Wnt signaling pathway by controlling the levels of cytoplasmic beta-catenin. Altered GSK3 beta expression has been described in various tumors, but to date, there are no studies evaluating its expression in models of oral carcinogenesis. Additionally, it is unknown whether the absence of galectin-3 regulates the expression of GSK3 beta. To this end, Gal3-deficient (Gal3(-/-)) and wild-type (Gal3(+/+)) male mice were treated with 4NQO for 16 weeks and sacrificed at week 16 and 32. The tongues were removed, processed, and stained with H&E to detect dysplasias and carcinomas. An immunohistochemical assay was performed to determine the level of P-GSK3 beta-Ser9 expression in both groups. Carcinomas were more prevalent in Gal3(+/+) than Gal3(-/-) mice (55.5% vs. 28.5%), but no statistical difference was reached. In the dysplasias, the proportion of cells positive for P-GSK3 beta-Ser9 was slightly higher in Gal3(+/+) than Gal3(-/-) mice (63% vs. 61%). In the carcinomas, a significant difference between Gal3(+/+) and Gal3(-/-) mice was found (74% vs. 59%; p=0.02). P-GSK3 beta-Ser9-positive cells slightly decreased from the progression of dysplasias to carcinomas in Gal3(-/-) mice (61% vs. 59%; p>0.05). However, a significant increase in P-GSK3 beta-Ser9 expression was observed from dysplasias to carcinomas in Gal3(+/+) mice (63% vs. 74%; p=0.01). In conclusion, these findings suggest that fully malignant transformation of the tongue epithelium is associated with increased P-GSK3 beta-Ser9 expression in Gal3(+/+) mice, but not in Gal3(-/-) mice.

Endurance exercise training increases APPL1 expression and improves insulin signaling in the hepatic tissue of diet-induced obese mice, independently of weight loss

Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. The protein kinase Akt plays a central role in the suppression of gluconeogenesis involving forkhead box O1 (Foxo1) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1a), and in the control of glycogen synthesis involving the glycogen synthase kinase beta (GSK3 beta) in the liver. It has been demonstrated that endosomal adaptor protein APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor, tribbles-related protein 3 (TRB3), improving the action of insulin in the liver. Here, we demonstrated that chronic exercise increased the basal levels and insulin-induced Akt serine phosphorylation in the liver of diet-induced obese mice. Endurance training was able to increase APPL1 expression and the interaction between APPL1 and Akt. Conversely, training reduced both TRB3 expression and TRB3 and Akt association. The positive effects of exercise on insulin action are reinforced by our findings that showed that trained mice presented an increase in Foxo1 phosphorylation and Foxo1/PGC-1a association, which was accompanied by a reduction in gluconeogenic gene expressions (PEPCK and G6Pase). Finally, exercised animals demonstrated increased at basal and insulin-induced GSK3 beta phosphorylation levels and glycogen content at 24?h after the last session of exercise. Our findings demonstrate that exercise increases insulin action, at least in part, through the enhancement of APPL1 and the reduction of TRB3 expression in the liver of obese mice, independently of weight loss. J. Cell. Physiol. 227: 29172926, 2012. (C) 2011 Wiley Periodicals, Inc.

Does Lithium Prevent Alzheimer's Disease?

Lithium salts have a well-established role in the treatment of major affective disorders. More recently, experimental and clinical studies have provided evidence that lithium may also exert neuroprotective effects. In animal and cell culture models, lithium has been shown to increase neuronal viability through a combination of mechanisms that includes the inhibition of apoptosis, regulation of autophagy, increased mitochondrial function, and synthesis of neurotrophic factors. In humans, lithium treatment has been associated with humoral and structural evidence of neuroprotection, such as increased expression of anti-apoptotic genes, inhibition of cellular oxidative stress, synthesis of brain-derived neurotrophic factor (BDNF), cortical thickening, increased grey matter density, and hippocampal enlargement. Recent studies addressing the inhibition of glycogen synthase kinase-3 beta (GSK3B) by lithium have further suggested the modification of biological cascades that pertain to the pathophysiology of Alzheimer's disease (AD). A recent placebo-controlled clinical trial in patients with amnestic mild cognitive impairment (MCI) showed that long-term lithium treatment may actually slow the progression of cognitive and functional deficits, and also attenuate Tau hyperphosphorylation in the MCI-AD continuum. Therefore, lithium treatment may yield disease-modifying effects in AD, both by the specific modification of its pathophysiology via inhibition of overactive GSK3B, and by the unspecific provision of neurotrophic and neuroprotective support. Although the clinical evidence available so far is promising, further experimentation and replication of the evidence in large scale clinical trials is still required to assess the benefit of lithium in the treatment or prevention of cognitive decline in the elderly.

Different approaches, one target: understanding cellular mechanisms of Parkinson's and Alzheimer's diseases

Neurodegenerative disorders are undoubtedly an increasing problem in the health sciences, given the increase of life expectancy and occasional vicious life style. Despite the fact that the mechanisms of such diseases are far from being completely understood, a large number of studies; that derive from both the basic science and clinical approaches have contributed substantial data in that direction. In this review, it is discussed several frontiers of basic research on Parkinson's and Alzheimer's diseases, in which research groups from three departments of the Institute of Biomedical Sciences of the University of Sao Paulo have been involved in a multidisciplinary effort. The main focus of the review involves the animal models that have been developed to study cellular and molecular aspects of those neurodegenerative diseases, including oxidative stress, insulin signaling and proteomic analyses, among others. We anticipate that this review will help the group determine future directions of joint research in the field and, more importantly, set the level of cooperation we plan to develop in collaboration with colleagues of the Nucleus for Applied Neuroscience Research that are mostly involved with clinical research in the same field.

Early and late neurodegeneration and memory disruption after intracerebroventricular streptozotocin

Glucose metabolism and insulin signaling disruptions in the brain have been proposed as a likely etiology of Alzheimer's disease. The aim of the present study was to investigate the time course of cognitive impairments induced by intracerebroventricular injection of streptozotocin (STZ) in rats and correlate them with the ensuing neurodegenerative process. Early and late effects of STZ were evaluated by using the reference and working memory versions of the Morris' water maze task and the evaluation of neurodegenerative markers by immunoblotting and the Fluoro-jade C histochemistry. The results revealed different types of behavioral and neurodegenerative responses, with distinct time courses. We observed an early disruption on the working memory as early as 3 h after STZ injections, which was followed by degenerative processes in the hippocampus at 1 and 15 days after STZ injections. Memory disruption increases over time and culminates with significant changes in amyloid-beta peptide and hyperphosphorylated Tau protein levels in distinct brain structures. These findings add information on the Alzheimer's disease-like STZ animal model and on the mechanisms underlying neurodegenerative processes. (C) 2012 Elsevier Inc. All rights reserved.

Intron 4 polymorphism of the endothelial nitric oxide synthase (eNOS) gene is associated with decreased NO production in a mercury-exposed population

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a potent vasodilator and plays a prominent role in regulating the cardiovascular system. Decreased basal NO release may predispose to cardiovascular diseases. Evidence suggests that the 27 nt repeat polymorphism of the intron 4 in the eNOS gene may regulate eNOS expression. On the other hand, some recent reports strongly suggest an association between methylmercury (MeHg) exposures and altered NO synthesis. In the present study, we investigate the contribution of the 27-pb tandem repeat polymorphism on nitric oxide production, which could enhance susceptibility to cardiovascular disease in the MeHg-exposed study population. Two-hundred-two participants (98 men and 104 women), all chronically exposed to MeHg through fish consumption were examined. Mean blood Hg concentration and nitrite plasma concentration were 50.5 +/- 35.4 mu g/L and 251.4 +/- 106.3 nM, respectively. Mean systolic and diastolic blood pressure were 120.1 +/- 19.4 mm Hg and 72.0 +/- 10.6 mm Hg, respectively. Mean body mass index was 24.5 +/- 4.3 kg/m(2) and the mean heart rate was 69.8 +/- 11.8 bpm. There were no significant differences in age, arterial blood pressure, body mass index or cardiac frequency between genotype groups (all P>0.05). However, we observed different nitrite concentrations in the genotypes groups, with lower nitrite levels for the 4a4a genotype carriers. Age, gender and the presence of intron 4 polymorphism contributed to nitrite reduction as a result of blood Hg concentration. Taken together, our results show that the 27 nt repeat polymorphism of the intron 4 in the eNOS gene increases susceptibility to cardiovascular diseases after MeHg exposure by modulating nitric oxide levels. (C) 2011 Elsevier B.V. All rights reserved.

Lack of beta(2)-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

beta(2)-adrenergic receptor (beta(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of beta(2)-AR activation are highly recognized, less is known about the impact of beta(2)-AR in endurance capacity. We presently used mice lacking beta(2)-AR [beta(2)-knockout (beta(2) KO)] to investigate the role of beta(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. beta(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, beta 2KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, beta 2KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in beta 2KO skeletal muscle. Altogether, these data provide evidence that disruption of beta(2)AR improves oxidative metabolism in skeletal muscle of beta 2KO mice and this is associated with increased exercise capacity.

Metabolic and functional effects of beta-hydroxy-beta-methylbutyrate (HMB) supplementation in skeletal muscle

Beta-hydroxy-beta-methylbutyrate (HMB) is a metabolite derived from leucine. The anti-catabolic effect of HMB is well documented but its effect upon skeletal muscle strength and fatigue is still uncertain. In the present study, male Wistar rats were supplemented with HMB (320 mg/kg per day) for 4 weeks. Placebo group received saline solution only. Muscle strength (twitch and tetanic force) and resistance to acute muscle fatigue of the gastrocnemius muscle were evaluated by direct electrical stimulation of the sciatic nerve. The content of ATP and glycogen in red and white portions of gastrocnemius muscle were also evaluated. The effect of HMB on citrate synthase (CS) activity was also investigated. Muscle tetanic force was increased by HMB supplementation. No change was observed in time to peak of contraction and relaxation time. Resistance to acute muscle fatigue during intense contractile activity was also improved after HMB supplementation. Glycogen content was increased in both white (by fivefold) and red (by fourfold) portions of gastrocnemius muscle. HMB supplementation also increased the ATP content in red (by twofold) and white (1.2-fold) portions of gastrocnemius muscle. CS activity was increased by twofold in red portion of gastrocnemius muscle. These results support the proposition that HMB supplementation have marked change in oxidative metabolism improving muscle strength generation and performance during intense contractions.

Association of polymorphisms of endothelial nitric oxide synthase (eNOS) gene with the risk of primary open angle glaucoma in a Brazilian population

The present study aimed to investigate the association of endothelial nitric oxide synthase (eNOS) gene polymorphisms with primary open angle glaucoma (POAG). We conducted a case-control study that included 90 patients with POAG and 127 healthy controls whose blood samples were genotyped for the functional polymorphisms T-786C and Glu298Asp of the eNOS gene by Taqman fluorescent allelic discrimination assay. The T-786C polymorphism was significantly associated as a risk factor for POAG among women (OR: 228; 95% CI: 1.11 to 4.70, p = 0.024) and marginally associated to the risk of POAG in the patients >= 52 years of age at diagnosis (OR: 2.11; 95% CI: 0.98 to 4.55, p = 0,055). However, these results was not confirmed after adjustments for gender, age, self-declared skin color, tobacco smoking and eNOS genotypes by multivariate logistic regression model (OR: 2.08; 95% CI: 0.87 to 5.01, p = 0.101 and OR: 2.20; 95% CI: 0.95 to 5.12, p = 0.067, respectively). The haplotype CG of T-786C and Glu298Asp showed a borderline association with risk of POAG in the overall analysis (OR: 1.76; 95% CI: 0.98 to 3.14, p = 0.055) and among women (OR: 2.02; 95% CI: 0.98 to 4.16, p = 0.052). Furthermore, the CG haplotype was significantly associated with the development of POAG for the age at diagnosis group >= 52 years (OR: 3.48; 95% CI: 1.54 to 7.84, p = 0.002). We suggested that haplotypes of the polymorphisms T-786C and Glu298Asp of eNOS may interact with gender and age in modulating the risk of POAG. (C) 2012 Elsevier B.V. All rights reserved.

Proposal of non-invasive experimental model to induce scoliosis in rats

Background: In the literature, there are several experimental models that induce scoliosis in rats; however, they make use of drugs or invasive interventions to generate a scoliotic curve. Objectives: To design and apply a non-invasive immobilization model to induce scoliosis in rats. Methods: Four-week old male Wistar rats (85 +/- 3.3 g) were divided into two groups: control (CG) and scoliosis (SG). The animals in the SG were immobilized by two vests (scapular and pelvic) made from polyvinyl chloride (PVC) and externally attached to each other by a retainer that regulated the scoliosis angle for twelve weeks with left convexity. After immobilization, the abdominal, intercostal, paravertebral, and pectoral muscles were collected for chemical and metabolic analyses. Radiographic reports were performed every 30 days over a 16-week period. Results: The model was effective in the induction of scoliosis, even 30 days after immobilization, with a stable angle of 28 +/- 5 degrees. The chemical and metabolic analyses showed a decrease (p<0.05) in the glycogenic reserves and in the relationship between DNA and total protein reserves of all the muscles analyzed in the scoliosis group, being lower (p<0.05) in the convex side. The values for the Homeostatic Model Assessment of Insulin Resistance indicated a resistance condition to insulin (p<0.05) in the scoliosis group (0.66 +/- 0.03), when compared to the control group (0.81 +/- 0.02). Conclusions: The scoliosis curvature remained stable 30 days after immobilization. The chemical and metabolic analyses suggest changes in muscular homeostasis during the induced scoliosis process.

Strain-specific polyketide synthase genes of Aspergillus niger

In silico comparison of 34 putative pks genes in Aspergillus niger strain CBS 513.88 versus A. niger strain ATCC 1015 genome revealed significant nucleotide identity (>95% covering a minimum of 99% of the gene sequence) for 31 of these genes (approximately 91%). A. niger CBS 513.88 harbors three putative pks genes (An01g01130, An11g05940, and An15g07920), for which nucleotide identity was not found in A. niger ATCC 1015. To compare the results of the in silico analysis with the in vivo situation, experimental data were obtained for a large number of A. niger strains obtained from different substrates and geographical regions. Three putative Os genes that were found to be variable between the two A. niger strains using bioinformatics tools were in fact strain-specific genes based on experimental data. The PCR amplification signals for the An01g01130, An11g05940, and An15g07920 pks genes were detected in only 97%, 71%, and 26% of the strains, respectively. Southern blot analyses confirmed the PCR data. Because one of the strain-specific pits genes (An15g07920) is located in a putative ochratoxin cluster, we focused our investigation on that region. We assessed the ochratoxin production capability of the 119 A. niger strains and found a positive association between the presence of this pia gene and the capability of the respective strain to produce ochratoxin. (C) 2012 Elsevier B.V. All rights reserved.

Knock out of neuronal nitric oxide synthase exacerbates intestinal ischemia/reperfusion injury in mice

Recent investigation of the intestine following ischemia and reperfusion (I/R) has revealed that nitric oxide synthase (NOS) neurons are more strongly affected than other neuron types. This implies that NO originating from NOS neurons contributes to neuronal damage. However, there is also evidence of the neuroprotective effects of NO. In this study, we compared the effects of I/R on the intestines of neuronal NOS knockout (nNOS(-/-)) mice and wild-type mice. I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the external muscle layers. Damage to the mucosa and muscle was more severe and greater infiltration by neutrophils occurred in the first 24 h in nNOS(-/-) mice. Immunohistochemistry for the contractile protein, alpha-smooth muscle actin, was used to evaluate muscle damage. Smooth muscle actin occurred in the majority of smooth muscle cells in the external musculature of normal mice but was absent from most cells and was reduced in the cytoplasm of other cells following I/R. The loss was greater in nNOS(-/-) mice. Basal contractile activity of the longitudinal muscle and contractile responses to nerve stimulation or a muscarinic agonist were reduced in regions subjected to I/R and the effects were greater in nNOS(-/-) mice. Reductions in responsiveness also occurred in regions of operated mice not subjected to I/R. This is attributed to post-operative ileus that is not significantly affected by knockout of nNOS. The results indicate that deleterious effects are greater in regions subjected to I/R in mice lacking nNOS compared with normal mice, implying that NO produced by nNOS has protective effects that outweigh any damaging effect of this free radical produced by enteric neurons.

The Diversity of Polyketide Synthase Genes from Sugarcane-Derived Fungi

The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase (PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26 shared sequence homology with beta-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase domains. A neighbour-joining phylogenetic analysis of the translated sequences could group the domains into previously established chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that, in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical ecology and likely biotechnological potential.

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-kappa B and upregulating endothelial nitric oxide synthase

de Souza ACCP, Volpini RA, Shimizu MH, Sanches TR, Camara NOS, Semedo P, Rodrigues CE, Seguro AC, Andrade L. Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting nuclear factor-kappa B and upregulating endothelial nitric oxide synthase. Am J Physiol Renal Physiol 302: F1045-F1054, 2012. First published January 11, 2012; doi:10.1152/ajprenal.00148.2011.-The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-kappa B activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP + EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-L-arginine methyl ester (L-NAME) simultaneously with EPO administration (CLP + EPO + L-NAME). A fifth group (CLP + EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP + EPO rats presented significantly higher inulin clearance than did CLP and CLP + EPO + L-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP + EPO rats; and inulin clearance was significantly higher in CLP + EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP + EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-alpha activation, NF-kappa B activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-kappa B downregulation.