A new amperometric biosensor for salicylate based on salicylate hydroxylase immobilized on polipyrrole film doped with hexacyanoferrate

An amperometric biosensor for salicylate detection was developed by immobilizing salicylate hydroxylase via glutaraldehyde onto a polypyrrole film doped with hexacyanoferrate, supported on a glassy carbon electrode surface. The sensor monitors the catechol produced in the enzymatic reaction on the film surface, at an applied potential of 150 mV vs. SCE. A [NADH]/[salicylate] ratio between 2 and 4 gave the best response. The biosensor presented the best performance in a solution with pH=7.4. The response time was about 40 s. A linear range of response was observed for salicylate concentrations between 1.0x10(-5) and 1.0x10(-4) mol l(-1) and the equation adjusted for this curve was I=(-0.04+/-0.01)+(11.4+/-0.2)[salicylate] with a correlation coefficient of 0.999 for n=6. The biosensor retains its activity for at least 10 days despite daily use. The results obtained using the biosensor for salicylate determination, in three different samples of antithermic drugs, presented a good correlation with the standard colorimetric method.

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries. © 2010 Elsevier Inc.

CYP21 gene mutations in Brazilian patients with 21-hydroxylase deficiency from the Amazon region

ABSTRACT: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (P450c21, CYP21) accounts for about 95% of all CAH cases. The incidence of CYP21 gene mutations has been extensively studied in the last years, but in Brazil it has been investigated only in Southeast Brazilian patients. This study is the first report on the distribution of CYP21 mutations in patients from the Amazon region. Direct sequencing of the CYP21 gene identified at least one mutation in 96% of the studied chromosomes. The most common mutations found were IVS2-13A/C > G (36%), Q318X (12%), V281L (12%), 1760_1761insT (9%), Cluster E6 (7%), and P30L (7%). The worldwide most common mutations were identified among patients from the Amazon region at frequencies that may be expected for a population resulting from the admixture of Europeans (predominantly Portuguese), African Blacks and Amerindians, in proportions that differ from those estimated for South Brazilian populations. Interethnic mixture may explain the differences in the frequencies of some mutations between Brazilian patients from the Amazon and from the Southeast of the country. However, the differences found may also be due to variation in the number of patients with the different clinical forms of 21-hydroxylase deficiency in the studies carried out so far.

Organisation and tyrosine hydroxylase and calretinin immunoreactivity in the main olfactory bulb of paca (cuniculus paca): a large caviomorph rodent

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of salicylate in blood serum using an amperometric biosensor based on salicylate hydroxylase immobilized in a polypyrrole-glutaraldehyde matrix

The use of an amperometric biosensor for the salicylate determination in blood serum is described. The biosensor is based on salicylate hydroxylase (EC 1.14.13.1) electropolymerized onto a glassy carbon-working electrode with polypyrrole and glutaraldehyde, to improve the biosensor lifetime. The hexacyanoferrate (II) was also incorporated to work as a redox mediator to minimize possible interferences. The salicylate is enzymatically converted to catechol, which is monitored amperometrically by its electrooxidation at +0.170 V versus SCE (saturated calomel electrode). Salicylate determination was carried out maintaining the ratio between β-NADH and salicylate at 4:1 (30°C). The amperometric response of the biosensor was linearly proportional to the salicylate concentration between 2.3 x 10-6 and 1.4 x 10-5 mol l- 1, in 0.1 mol l-1 phosphate buffer (pH 7.8), containing 0.1 mol l-1 KCl and 5.0 x 10-4 mol l-1 Na2H2EDTA, as supporting electrolyte. The recovery studies, in the presence of several interfering compounds, showed recoveries between 96.4 and 104.8%. The useful lifetime of the biosensor in the concentration range evaluated was at least 40 days, in continuous use. Blood serum samples analyzed by this biosensor showed a good correlation compared to the spectrophotometric method (Trinder) used as reference, presenting relative deviations lower than 7.0%. (C) 2000 Elsevier Science B.V.

Impact of Glucocorticoid Receptor Gene Polymorphisms on the Metabolic Profile of Adult Patients with the Classical Form of 21-Hydroxylase Deficiency

Background: CAH patients have an increased risk of cardiovascular disease, and it remains unknown if lifelong glucocorticoid (GC) treatment is a contributing factor. In the general population, glucocorticoid receptor gene (NR3C1) polymorphisms are associated with an adverse metabolic profile. Our aim was to analyze the association between the NR3C1 polymorphisms and the metabolic profile of CAH patients. Methodology: Sixty-eight adult patients (34SV/34SW) with a mean age of 28.4 +/- 9 years received dexamethasone (mean 0.27 +/- 0.11 mg/day) to obtain normal androgen levels. SW patients also received fludrocortisone (50 mu g/day). Metabolic syndrome (MetS) was defined by the NCEP ATPIII criteria and obesity by BMI >= 30 kg/m(2). NR3C1 alleles were genotyped, and association analyses with phenotype were carried out with Chi-square, t-test and regression analysis. Results: Obesity and MetS were observed in 23.5% and 7.3% of patients, respectively, and were not correlated with GC doses and treatment duration. BMI was positively correlated with blood pressure (BP), triglycerides (TG), LDL-c levels and HOMA-IR and inversely correlated with HDL-c levels. BclI and A3669G variants were found in 26.4% and 9.6% of alleles, respectively. Heterozygotes for the BclI polymorphism presented with higher BMI (29 kg/m(2) +/- 5.3 vs. 26 kg/m(2) +/- 5.3, respectively) and waist circumference (89 cm +/- 12.7 vs. 81 cm +/- 13, respectively) compared to wild-type subjects. Hypertension was found in 12% of patients and heterozygotes for the BclI polymorphism presented higher systolic BP than wild type subjects. Low HDL-c and high TG levels were identified in 30% and 10% of patients, respectively, and were not associated with the NR3C1 polymorphisms. A3669G carriers and non-carriers did not differ. Conclusion: In addition to GC therapy, the BclI GR variant might play an important role in obesity susceptibility in CAH patients. Genotyping of GR polymorphisms could result in the identification of a subgroup at risk patients, allowing for the establishment of personalized treatment and the avoidance of long-term adverse consequences.

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 mu g/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-alpha-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-alpha-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-alpha-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'. (C) 2011 Elsevier Ltd. All rights reserved.

Congenital Adrenal Hyperplasia Due to 21 Hydroxylase Deficiency: From Birth to Adulthood

The most frequent form of congenital adrenal hyperplasia (CAH) is steroid 21-hydroxylase deficiency, accounting for more than 90% of cases. Affected patients cannot synthesize cortisol efficiently. Thus the adrenal cortex is stimulated by corticotropin (ACTH) and overproduces cortisol precursors. Some precursors are diverted to sex hormone biosynthesis, causing signs of androgen excess including ambiguous genitalia in newborn females and rapid postnatal growth in both sexes. In the most severe "salt wasting" form of CAH (similar to 75% of severe or "classic" cases), concomitant aldosterone deficiency may lead to salt wasting with consequent failure to thrive, hypovolemia, and shock. Newborn screening minimizes delays in diagnosis, especially in males, and reduces morbidity and mortality from adrenal crises. CAH is a recessive disorder caused by mutations in the CYP21 (CYP21A2) gene, most of which arise from recombination between CYP21 and a nearby pseudogene, CYP21P (CYP21A1P). Phenotype is generally correlated with genotype. Classic CAH patients require chronic glucocorticoid treatment at the lowest dose that adequately suppresses adrenal androgens and maintains normal growth and weight gain, and most require mineralocorticoid (fludrocortisone). Transition of care of older patients to adult physicians should be planned in advance as a structured, ongoing process.

Sintesi di 4-bromo-pirazoli quali precursori di sistemi eterociclici condensati. Sintesi della 3a,4,5,6,7,7a-esaidro-N-metil-1H-1,4,6-(epietano-1,1,2-triil)indeno-4,8-dicarbossimmide e di composti relazionati tramite reazione Diels-Alder intramolecolare.

Il progetto di tesi specialistica svolto durante questo anno accademico si è suddiviso in due parti: un primo periodo, da settembre 2010 a gennaio 2011, presso il dipartimento di Chimica Organica “A. Mangini” della Facoltà di Chimica Industriale dell’Università di Bologna e un secondo periodo in Spagna, da marzo ad agosto 2011, presso la Unitat de Química Farmacèutica de la Facultat de Farmàcia de la Universitat de Barcelona. Nel primo periodo a Bologna mi sono occupato della sintesi di 4-bromo-pirazoli da utilizzare come precursori di composti eterociclici condensati. Inizialmente è stato sintetizzato un pirazolo 1,3,5-trisostituito tramite cicloaddizione 1,3-dipolare tra un acetilene e una nitril immina generata in situ da un idrazonoil cloruro. Il pirazolo è stato poi bromurato facendo uno screening di diversi agenti bromuranti e condizioni di reazione per ottenere la migliore resa e chemoselettività. Infine è stata studiata la ciclizzazione intramolecolare del prodotto bromurato tramite reazione di cross-coupling catalizzata da metalli di transizione. Nel secondo periodo a Barcellona mi sono occupato della sintesi di dicarbossimmidi tricicliche con struttura a gabbia con il fine di creare alcheni altamente piramidalizzati e di studiarne la dimerizzazione ad un derivato del dodecaedrano. La strategia sintetica è stata impostata utilizzando come reagente di partenza una semplice succinimmide per giungere, dopo numerosi passaggi, al precursore del prodotto triciclico, del quale è stata studiata la ciclizzazione tramite reazione Diels-Alder intramolecolare.

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor in dental pulp-derived cells

Introduction: Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp–derived cells. Methods: To test the response of dental pulp–derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, 3[H]thymidine, and 3[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). Results: We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp–derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. Conclusions: These results show that PHD inhibitors can stimulate VEGF production of dental pulp–derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Acute Encephalopathy with Unilateral Cortical-Subcortical Lesions in Two Unrelated Kindreds Treated with Glucocorticoids Prenatally for Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency: Established Facts and Novel Insight

Background: Prenatal glucocorticoid (GC) treatment of the female fetus with 21-hydroxylase deficiency (21-OHD) may prevent genital virilization and androgen effects on the brain, but prenatal GC therapy is controversial because of possible adverse effects on fetal programming, the cardiovascular system and the brain. Case Reports: We report 2 patients with congenital adrenal hyperplasia (CAH) due to 21-OHD who were treated prenatally with dexamethasone, suffered from an acute encephalopathy and showed focal and multifocal cortical and subcortical diffusion restrictions in early MRI and signs of permanent alterations in the follow-up neuroimaging studies. Both patients recovered from the acute episode. Whereas the first patient recovered without neurological sequelae the second patient showed hemianopsia and spastic hemiplegia in the neurological follow-up examination. Conclusion: These are 2 children with CAH, both treated prenatally with high doses of dexamethasone to prevent virilization. The question arises whether prenatal high-dose GC treatment in patients with CAH might represent a risk factor for brain lesions in later life. Adverse effects/events should be reported systematically in patients undergoing prenatal GC treatment and long-term follow-up studies involving risk factors for cerebrovascular disease should be performed.