The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo-and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.
Background: Plasma free and urinary metanephrines are recognized biomarkers for the assessment of pheochromocytoma. Plasma total metanephrines with a long half-life may represent another useful biomarker. Objective: The aim of this study is to evaluate the diagnostic performances of plasma total metanephrines alone or combined with free metanephrines and fractionated 24-h urinary metanephrines. Methods: A retrospective, case-control diagnostic test study was conducted between 1999 and 2007 in two university hospitals in Switzerland and two institutions in France. The patients included 46 cases with histologically proven pheochromocytoma, and 181 controls suspected of tumor with negative investigations and 3-year follow-up. None had renal dysfunction. Sensitivity and specificity were compared after expressing each measurement result as a ratio over its upper reference limit, adding the ratios of normetanephrine and metanephrine, and defining cut-off values of 1 or 2 for this sum. Results: Applying a cut-off value of 1, plasma free and total metanephrines and urinary fractionated metanephrines had similar sensitivities of 96% (95% confidence interval, 86-99%), 95% (85-99%), and 95% (84-99%) along with similar specificities of 89% (83-94%), 91% (84-95%), and 86% (80-91%). A cut-off of 2 for the sum of ratios over reference limit improves the specificity, and it can be used for a confirmation test based on another biomarker taken among the three biomarkers. Conclusion: All three metanephrine-based tests perform equivalently for diagnosing pheochromocytoma in the absence of renal insufficiency, and can be conveniently associated two by two for confirming/excluding tumor.
The contribution of neuropeptide Y (NPY), deriving from adrenal medulla, to the adrenosympathetic tone is unknown. We found that in response to NPY, primary cultures of mouse adrenal chromaffin cells secreted catecholamine, and that this effect was abolished in cultures from NPY Y1 receptor knockout mice (Y1؊͞؊). Compared with wild-type mice (Y1؉͞؉), the adrenal content and constitutive release of catecholamine were increased in chromaffin cells from Y 1؊͞؊ mice. In resting animals, catecholamine plasma concentrations were higher in Y1؊͞؊ mice. Comparing the adrenal glands of both genotypes, no differences were observed in the area of the medulla, cortex, and X zone. The high turnover of adrenal catecholamine in Y 1؊͞؊ mice was explained by the enhancement of tyrosine hydroxylase (TH) activity, although no change in the affinity of the enzyme was observed. The molecular interaction between the Y 1 receptor and TH was demonstrated by the fact that NPY markedly inhibited the forskolin-induced luciferin activity in Y 1 receptor-expressing SK-N-MC cells transfected with a TH promoter sequence. We propose that NPY controls the release and synthesis of catecholamine from the adrenal medulla and consequently contributes to the sympathoadrenal tone. (3,4). NPY is an important neurotransmitter of the sympathetic function that potentiates the catecholamine vasoconstrictor activity through the Y 1 receptor and exerts prejunctional inhibitory effects on NE release from the sympathetic nerve endings of the heart through the Y 2 receptor (5). In addition, the nerve terminals of parasympathetic neurons in the mouse heart possess Y 2 receptors, which, when activated, reduce acetylcholine release, also causing an inhibition of the parasympathetic nervous system (6). We have shown that NPY Y 1 knockout mice (Y 1 Ϫ͞Ϫ) lose their ability to potentiate NE-induced vasoconstriction and have normal blood pressure, probably indicating a minor role of NPY in the maintenance of blood pressure homeostasis (7). Recently, these mice were investigated for their cardiac sympathovagal balance in baseline conditions and during an acute social challenge. Reduced somatomotor activity during nonsocial challenges, lower heart rate in baseline conditions, and larger heart rate responsiveness during social defeat were reported (8). Besides its presence in nerve endings, NPY is produced by chromaffin cells of adrenal medulla of different species, including human (9). The mouse has higher adrenal NPY content than rat, pig, or humans (10, 11). The effect of NPY on the adrenal medulla is controversial. NPY stimulated catecholamine release from intact rat adrenal capsular tissue (12), although an inhibitory effect of NPY on catecholamine secretion in rat adrenomedullary primary cell cultures was also observed (13). Moreover, there is a weak inhibitory effect of NPY on NE and epinephrine (EP) release from bovine chromaffin cells, evoked by addition of a cholinergic agonist (14, 15). However, depending on the experimental conditions, conflicting results wer...
Epinephrine (E) and norepinephrine (NE) play a major role in regulating metabolism and cardiovascular physiology. Both are secreted in response to stress and their measurement in plasma allows the study of sympathoadrenal function. Several studies investigating sympathoadrenal physiology are conducted using mice. Review of the literature revealed that basal mouse NE and E plasma concentrations range within 4-140 nM depending on the blood sampling method. Such variability doesn't allow study comparison and may conceal catecholamine variations in response to stress. Therefore, our aim was to determine a reliable sampling method to measure mouse plasma catecholamine concentrations. Results showed that arterial catheterization is the most accurate sampling method: E and NE basal levels were similar to those found in humans (1.1€0.3 nM and 4.1€0.5 nM, respectively). Retro-orbital bleeding led to analogous results. On the contrary, decapitation was stressful for mice and consequently NE and E concentrations were high (24.6€2.7 nM and 27.3€3.8 nM, respectively). These different bleeding methods were compared in terms of their ability to detect sympathoadrenal system stimulation (cold-pressure test). With catheter and retroorbital samplings the expected increase in NE and E levels was easily perceived. In contrast, with decapitation no significant change in E was detected. In conclusion, arterial-catheter and retro-orbital blood sampling methods appear to be the most accurate procedures for studying the sympathetic nervous system in mice in both unstressed and stressed conditions.
The aim of the present work was to study the effect of angiotensin II (Ang II) on catecholamines and neuropeptide Y (NPY) release in primary cultures of human adrenal chromaffin cells. Ang II stimulates norepinephrine (NE), epinephrine (EP) and NPY release from perifused chromaffin cells by 3-, 2-and 12-fold, respectively. The NPY release is more sustained than that of catecholamines. We found that the receptor-AT 2 agonist, T 2 -(Ang II 4 -8) 2 has no effect on NE, EP and NPY release from chromaffin cells. We further showed that Ang II increases intracellular Ca 2 + concentration ([Ca 2 + ] i ). The selective AT 1 -receptor antagonist Candesartan blocked [Ca 2 + ] i increase by Ang II, while T 2 -(Ang II 4 -8) 2 was ineffective. These findings demonstrate that AT 1 stimulation induces catecholamine secretion from human adrenal chromaffin cells probably by raising cytosolic calcium. D
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