Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.

Loss of the Ca2+/calmodulin-dependent protein kinase type IV in dopaminoceptive neurons enhances behavioral effects of cocaine

The persistent nature of addiction has been associated with activity-induced plasticity of neurons within the striatum and nucleus accumbens (NAc). To identify the molecular processes leading to these adaptations, we performed Cre/loxP-mediated genetic ablations of two key regulators of gene expression in response to activity, the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) and its postulated main target, the cAMP-responsive element binding protein (CREB). We found that acute cocaine-induced gene expression in the striatum was largely unaffected by the loss of CaMKIV. On the behavioral level, mice lacking CaMKIV in dopaminoceptive neurons displayed increased sensitivity to cocaine as evidenced by augmented expression of locomotor sensitization and enhanced conditioned place preference and reinstatement after extinction. However, the loss of CREB in the forebrain had no effect on either of these behaviors, even though it robustly blunted acute cocaine-induced transcription. To test the relevance of these observations for addiction in humans, we performed an association study of CAMK4 and CREB promoter polymorphisms with cocaine addiction in a large sample of addicts. We found that a single nucleotide polymorphism in the CAMK4 promoter was significantly associated with cocaine addiction, whereas variations in the CREB promoter regions did not correlate with drug abuse. These findings reveal a critical role for CaMKIV in the development and persistence of cocaine-induced behaviors, through mechanisms dissociated from acute effects on gene expression and CREB-dependent transcription.

Circulating levels of brain-derived neurotrophic factor: correlation with mood, cognition and motor function

Brain-derived neurotrophic factor (BDNF) is the most widely distributed neurotrophin in the CNS, where it plays several pivotal roles in synaptic plasticity and neuronal survival. As a consequence, BDNF has become a key target in the physiopathology of several neurological and psychiatric diseases. Recent studies have consistently reported altered levels of BDNF in the circulation (i.e., serum or plasma) of patients with major depression, bipolar disorder, Alzheimer`s disease, Huntington`s disease and Parkinson`s disease. Correlations between serum BDNF levels and affective, cognitive and motor symptoms have also been described. BDNF appears to be an unspecific biomarker of neuropsychiatric disorders characterized by neurodegenerative changes.

High-Resolution Genomic Mapping Reveals Consistent Amplification of the Fibroblast Growth Factor Receptor Substrate 2 Gene in Well-Differentiated and Dedifferentiated Liposarcoma

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31 CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) ""single nucleotide polymorphism""/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at 1436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma. (C) 2011 Wiley-Liss, Inc.

Influence of the Fibroblast Growth Factor Receptor 4 Expression and the G388R Functional Polymorphism on Cushing`s Disease Outcome

Context: Abnormal FGFR4 expression has been detected in pituitary tumors, especially in larger and invasive adenomas. In addition, the FGFR4 functional polymorphism G388R has been associated with poor outcome in several human malignancies. Then, we hypothesized that FGFR4 expression and genotype could be markers of adverse outcome of Cushing`s disease after transsphenoidal surgery. Objectives: The objective was to investigate whether there is an association between the postoperative outcome of Cushing`s disease (remission/recurrence) and the FGFR4 G388R genotype or the FGFR4 expression in corticotrophinomas. Design and Patients: Clinical, hormonal, and pathological data of 76 patients who underwent the first transsphenoidal surgery were retrospectively reviewed. All patients were genotyped for G388R polymorphism. FGFR4 expression was assessed by real-time PCR in 18 corticotrophinomas. Main Outcome Measures: The outcome measures included the FGFR4 G388R genotype and FGFR4 expression in postoperative remission and recurrence of Cushing`s disease. Results: Homozygosis for FGFR4 glycine (Gly(388)) allele was associated with reduced disease-free survival, in the univariate analysis (hazard ratio of 6.91; 95% confidence interval of 1.14-11.26; P = 0.028). Male gender (P = 0.036), lack of pathology confirmation (P = 0.009), and cortisol levels more than 2 mu g/dl in the early postoperative period (P < 0.001) were also significant predictors of Cushing`s disease recurrence in the univariate analysis. FGFR4 overexpression was found in 44% of the corticotrophinomas, and it was associated with lower postoperative remission rate (P = 0.009). Conclusions: Our data suggest that homozygosis for FGFR4 Gly(388) allele and FGFR4 overexpression are associated with higher frequency of postoperative recurrence and persistence of Cushing`s disease, respectively. (J Clin Endocrinol Metab 95: E271-E279, 2010)

Rapid antidepressant changes with sleep deprivation in major depressive disorder are associated with changes in vascular endothelial growth factor (VEGF): A pilot study

While conventional antidepressants benefit many patients with major depressive disorder (MDD), as much as eight to 12 weeks can elapse before significant improvements in depressive symptoms are seen. Treatments that act more rapidly in MDD are urgently needed. Sleep deprivation (SD) has been shown to produce a rapid antidepressant response within one day in 50-60% of patients with MDD; thus, identifying its antidepressant mechanism may contribute to the development of antidepressants that act more rapidly. The present study evaluated the effects of 39 h of SD on mood, as well as on plasma levels of brain derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in patients with MDD. After a drug-free period of at least two weeks, 11 patients (6 males, 5 females; ages 25-62) who met DSM-IV criteria for MDD underwent total SD. Plasma samples for BDNF and VEGF assays were collected on Days 1 (baseline) and 2. The six-item Hamilton Rating Scale for Depression (HAMD-6) was the primary outcome measure. HAMD-6 scores decreased significantly after SD (Day 2). SD was negatively correlated with change in HAMD-6 score and change in VEGF levels, indicating that as depression scores decreased following SD, VEGF plasma levels increased. In contrast, SD did not alter plasma BDNF concentrations, nor was an association found between BDNF levels and clinical improvement on the HAMD-6. These results suggest that SD is associated with mood-related changes in plasma VEGF levels, but not plasma BDNF levels. Further studies using larger sample sizes are needed to confirm these preliminary findings. Published by Elsevier Inc.

Monoclonal anti-vascular endothelial growth factor antibody reduces fluid volume in an experimental model of inflammatory pleural effusion

Background and objective: Vascular endothelial growth factor (VEGF) is known to increase vascular permeability and promote angiogenesis. It is expressed in most types of pleural effusions. However, the exact role of VEGF in the development of pleural effusions has yet to be determined. The anti-VEGF mAb, bevacizumab, has been used in the treatment of cancer to reduce local angiogenesis and tumour progression. This study describes the acute effects of VEGF blockade on the expression of inflammatory cytokines and pleural fluid accumulation. Methods: One hundred and twelve New Zealand rabbits received intrapleural injections of either talc or silver nitrate. In each group, half the animals received an intravenous injection of bevacizumab, 30 min before the intrapleural agent was administered. Five animals from each subgroup were sacrificed 1, 2, 3, 4 or 7 days after the procedure. Twelve rabbits were used to evaluate vascular permeability using Evans`s blue dye. Pleural fluid volume and cytokines were quantified. Results: Animals pretreated with anti-VEGF antibody showed significant reductions in pleural fluid volumes after talc or silver nitrate injection. IL-8 levels, vascular permeability and macroscopic pleural adhesion scores were also reduced in the groups that received bevacizumab. Conclusions: This study showed that bevacizumab interferes in the acute phase of pleural inflammation induced by silver nitrate or talc, reinforcing the role of VEGF as a key mediator in the production of pleural effusions. The results also suggest that bevacizumab should probably be avoided in patients requiring pleurodesis.

Fetal hemodynamic changes following maternal betamethasone administration in pregnancies with fetal growth restriction and absent end-diastolic flow in the umbilical artery

Objectives. To examine the effects of betamethasone administration on umbilical artery (UA), middle cerebral artery (MCA) and ductus venosus (DV) Doppler flow. Design. Longitudinal prospective study. Setting: Fetal Surveillance Unit, Department of Obstetrics and Gynecology, University of Sao Paulo, Sao Paulo, Brazil. Population. Thirty-two singleton pregnancies complicated by fetal growth restriction with absent end-diastolic flow in the UA. Methods. Pulsatility index (PI) of the UA, MCA and DV was measured from 26 to 34 weeks prior to and within 24 or 48 hours after starting betamethasone treatment course. Analysis of variance for repeated measures was used to determine the changes in the fetal hemodynamic Doppler flow following maternal corticosteroid administration. Main outcome measures. Improvement of UA-PI within 24 hours and DV-PIV (venous pulsatility) within 48 hours from the first betamethasone dose. Results. Mean gestational age at delivery was 29.3 (1.8) weeks and birthweight was 806.6 (228.2) g. A reduction in the UA-PI was observed in 29 (90.6%) cases, with return of end-diastolic flow in 22 (68.7%). The mean UA-PI were 2.84 (0.52) before corticosteroid administration, 2.07 (0.56) within 24 hours and 2.42 (0.75) after 48 hours, with a significant difference along the evaluations (p0.001). No significant changes in the MCA Doppler were observed. DV-PIV decreased from 1.06 (0.23) prior corticosteroids administration to 0.73 (0.16) within 24 hours and 0.70 (0.19) after 48 hours (p0.001). Conclusions. There was reduction in the umbilical artery and in the DV pulsatility indices within 24 hours from betamethasone administration that was maintained up to 48 hours.

Analysis of Craniofacial and Extremity Growth in Patients with Growth Hormone Deficiency during Growth Hormone Therapy

Background/Aims: There are many controversies regarding side effects on craniofacial and extremity growth due to growth hormone ( GH) treatment. Our aim was to estimate GH action on craniofacial development and extremity growth in GH-deficient patients. Methods: Twenty patients with GH deficiency with a chronological age ranging from 4.6 to 24.3 years (bone age from 1.5 to 13 years) were divided in 2 groups: group 1 (n = 6), naive to GH treatment, and group 2 (n = 14), ongoing GH treatment for 2-11 years. GH doses (0.1 -0.15 U/kg/day) were adjusted to maintain insulin-like growth factor 1 and insulin-like growth factor binding protein 3 levels within the normal range. Anthropometric measurements, cephalometric analyses and facial photographs to verify profile and harmony were performed annually for at least 3 years. Results: Two patients with a disharmonious profile due to mandibular growth attained harmony, and none of them developed facial disharmony. Increased hand or foot size (>P97) was observed in 2 female patients and in 4 patients (1 female), respectively, both not correlated with GH treatment duration and increased levels of insulin-like growth factor 1. Conclusions: GH treatment with standard doses in GH-deficient patients can improve the facial profile in retrognathic patients and does not lead to facial disharmony although extremity growth, mainly involving the feet, can occur. Copyright (C) 2009 S. Karger AG, Basel

High prevalence of pituitary magnetic resonance abnormalities and gene mutations in a cohort of Brazilian children with growth hormone deficiency and response to treatment

Data were retrospectively collected from 69 Brazilian patients (45 boys) with growth hormone deficiency (GHD) who received exogenous growth hormone (GH) for a median duration of 4 years (range 1-13 years). Forty-two patients had multiple pituitary hormone deficiencies and 27 had isolated GHD. Peak GH was <7 ng/ml (IRMA) or <3.2 ng/ml (IFMA) after two stimulation tests.. Therapy was started at median age of 10.0 years (range 2.2-21.6 years), bone age of 5.8 years (0.5-13.5 years) and height standard deviation score -4.4 (range -9.3 to -1.6). MRI revealed pituitary abnormalities in 87% of patients. Homozygous mutations in PROP-1, GHRH-R, GH-1 or HESX-1 genes were found in 12 patients. Mean height velocities were 3.3 pretreatment and 10.3, 7.8, 7.4 and 6.4 cm/yr, respectively, during 1-4 years of treatment with GH. In conclusion, the high prevalence (96%) of genetic and/or pituitary abnormalities probably reflects the stringent diagnostic criteria used, and GH replacement resulted in significant catch-up growth.

Trans-resveratrol inhibits early blood vessel formation (Vasculogenesis) without impairment of embryonic growth

Resveratrol is a stilbene compound found in grapes and other sources. In this study we examined the effects of trans-resveratrol (4.38-438 mu M/implant) in the vasculogenesis of yolk-sac membranes and its capacity to improve chick embryo growth. High concentrations of the stilbene (43.8-438 mu M) significantly inhibited early vessel formation, decreasing the percentage vitelline vessels of 3.5-day embryos by 50% compared to the control. In addition, basic fibroblast growth factor-stimulated vasculogenesis (140% of vessels as compared to control) was partially reversed by t-resveratrol (35% of inhibition) and treatments with cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) as well a protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate) decreased the vessel number to 60%, 50%, and 44%, respectively. Treatments with t-resveratrol (4.38-43.8 mu M/implant) significantly increased the body length of embryos incubated in vitro uncoupled from any impairment in the body shape or detectable embryotoxic effect. We suggest that the antivasculogenic activity and the enhancement in embryonic growth promoted by non acute treatments with t-resveratrol were, at least in part, due to PKC inhibition. We suggest that t-resveratrol can be usable not only as a reliable functional nutriment, but also is useful for the development of prophylactic and/or therapeutic agents for treatment of angiogenic-degenerative diseases.

Mandibular appliance modulates condylar growth through integrins

Functional orthopedic therapy corrects growth discrepancies between the maxilla and mandible, possibly through postural changes in the musculature and modulation of the mandibular condylar cartilage growth. Using Wistar rats, we tested the hypothesis that chondrocytes respond to forces generated by a mandibular propulsor appliance by changes in gene expression, and that integrins are important mediators in this response. Immunohistochemical analyses demonstrated that the use of the appliance for different periods of time modulated the expression of fibronectin, alpha 5 and alpha v integrin subunits, as well as cell proliferation in the cartilage. In vitro, cyclic distension of condylar cartilage-derived cells increased fibronectin mRNA, as well as Insulinlike Growth Factor-I and II mRNA and cell proliferation. A peptide containing the Arginine-Glycine-Asparagine sequence (RGD), the main cell-binding sequence in fibronectin, blocked almost all these effects, confirming that force itself modulates the growth of the rat condylar cartilage, and that RGD-binding integrins participate in mechanotransduction.

Novel inactivating mutations in the GH secretagogue receptor gene in patients with constitutional delay of growth and puberty

Background: A limited number of mutations in the GH secretagogue receptor gene (GHSR) have been described in patients with short stature. Objective: To analyze GHSR in idiopathic short stature (ISS) children including a subgroup of constitutional delay of growth and puberty (CDGP) patients. Subjects and methods: The GHSR coding region was directly sequenced in 96 independent patients with ISS, 31 of them with CDGP, in 150 adults, and in 197 children with normal stature. The pharmacological consequences of GHSR non-synonymous variations were established using in vitro cell-based assays. Results: Five different heterozygous point variations in GHSR were identified (c.-6 G>C, c.251G>T (p.Ser84Ile), c.505G>A (p.Ala169Thr), c.545 T>C (p.Val182Ala), and c.1072G>A (p.Ala358Thr)), all in patients with CDGP. Neither these allelic variants nor any other mutations were found in 694 alleles from controls. Functional studies revealed that two of these variations (p.Ser84Ile and p. Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. These mutations were identified in two female patients with CDGP (at the age of 13 years, their height SDS were -2.4 and -2.3). Both patients had normal progression of puberty and reached normal adult height (height SDS of -0.7 and -1.4) without treatment. Conclusion: This is the first report of GHSR mutations in patients with CDGP. Our data raise the intriguing possibility that abnormalities in ghrelin receptor function may influence the phenotype of individuals with CDGP.

Antenatal steroid and tracheal occlusion restore vascular endothelial growth factor receptors in congenital diaphragmatic hernia rat model

OBJECTIVE: Investigate the effects of antenatal steroids and tracheal occlusion on pulmonary expression of vascular endothelial growth factor receptors in rats with nitrofen-induced congenital diaphragmatic hernia. STUDY DESIGN: Fetuses were exposed to nitrofen at embryonic day 9.5. Subgroups received dexamethasone or were operated on for tracheal occlusion, or received combined treatment. Morphologic variables were recorded. To analyze vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, we performed Western blotting and immunohistochemistry. Morphologic variables were analyzed by analysis of variance and immunohistochemistry by Kruskal-Wallis test. RESULTS: Congenital diaphragmatic hernia decreased body weight, total lung weight, and lung-to-body weight ratio. Tracheal occlusion increased total lung weight and lung-to-body weight ratio (P < .05). Fetuses with congenital diaphragmatic hernia had reduced vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, whereas steroids and tracheal occlusion increased their expression. Combined treatment increased expression of receptors, but had no additive effect. CONCLUSION: Vascular endothelial growth factor signaling disruption may be associated with pulmonary hypertension in congenital diaphragmatic hernia. Tracheal occlusion and steroids provide a pathway for restoring expression of vascular endothelial growth factor receptors.

Assessment of the Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta in a Rat Model of Intrauterine Growth Restriction

Objective: To investigate glomerular development and expression of insulin and insulin-like growth factor receptors in an experimental model of intrauterine growth restriction (IUGR). Material and Methods: We studied three groups of Sprague-Dawley fetuses: IUGR - restricted by ligation of the right uterine artery; C-IUGR - left horn controls, and EC - external controls (non-manipulated). Body and organs were weighed, and glomerular number and volume were analyzed. Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta was analyzed in liver, intestine and kidneys by immunoblotting. Results: Organ/body weight ratios were similar. In IUGR, glomerular number and volume were increased compared to C-IUGR and EC (p < 0.001). In the IUGR liver, increases were found in IGF-IR beta compared to C-IUGR and EC; IR beta compared to EC, and IRS-2 compared to C-IUGR. However, decreases in IR beta were noted in IUGR compared to C-IUGR; IRS-1 compared to C-IUGR and EC, and IRS-2 compared to EC. In IUGR intestine, increases were detected in IR beta, IRS-1 and IGF-IR beta compared to C-IUGR and EC. In IUGR kidneys, increases were observed in IR beta and IGF-IR beta compared to C-IUGR and EC, and IRS-1 compared to EC. Decreased IRS-2 in the intestine and kidney were noticed in IUGR compared to C-IUGR and EC. Conclusion: IUGR fetuses had less glomeruli and alterations in insulin receptors, which may be associated with an increased risk of disease occurrence in adulthood. Copyright (C) 2010 S. Karger AG, Basel