A novel mammalian adenylyl cyclase was identified by reverse transcription-polymerase chain reaction amplification using degenerate primers based on a conserved region of previously described adenylyl cyclases (Premont, R. T. (1994) Methods Enzymol. 238, 116-127). The full-length cDNA sequence obtained from mouse brain predicts a 1353-amino acid protein possessing a 12-membrane span topology, and containing two regions of high similarity with the catalytic domains of adenylyl cyclases. Comparison of this novel adenylyl cyclase with the eight previously described mammalian enzymes indicates that this type 9 adenylyl cyclase sequence is the most divergent, defining a sixth distinct subclass of mammalian adenylyl cyclases. The AC9 gene has been localized to human chromosome band 16p13.3-13.2. The 8.5-kb mRNA encoding the type 9 adenylyl cyclase is widely distributed, being readily detected in all tissues tested, and is found at very high levels in skeletal muscle and brain. AC9 mRNA is found throughout rat brain but is particularly abundant in hippocampus, cerebellum, and neocortex. An antiserum directed against the carboxyl terminus of the type 9 adenylyl cyclase detects native and expressed recombinant AC9 protein in tissue and cell membranes. Levels of the AC9 protein are highest in mouse brain membranes. Characterization of expressed recombinant AC9 reveals that the protein is a functional adenylyl cyclase that is stimulated by Mg2+, forskolin, and mutationally activated Gsalpha. AC9 activity is not affected by Ca2+/calmodulin or by G protein betagamma-subunits. Thus AC9 represents a functional G protein-regulated adenylyl cyclase found in brain and in most somatic tissues.
Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%o) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calciumcalmodulin complex. These findings suggest (i) the involvement of ACI acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of ACi in the rat pineal gland.In living organisms, development, maturation, or reproduction usually undergo circadian or seasonal variations. In dictating the adaptative response of the organism to this temporal program, the pineal gland plays a key role, functioning as a neuroendocrine transducer. During the night-day cycle, input information from the retina is perceived at the pinealocyte membrane level as a rhythmic nocturnal norepinephrine (NE) signal, via a multisynaptic modulatory pathway (1). This neural information is then converted into a hormonal message, the nocturnal melatonin production and release, a phenomenon mediated essentially by the cyclic AMP pathway (2-4).The nocturnal rise in cyclic AMP amount (5) and the subsequent increase in cyclic AMP-responsive elementbinding protein (CREB) phosphorylation (6) account for the nocturnal profile of melatonin production, by stimulation of both the transcription and the activity of serotonin Nacetyltransferase (NAT), the rate limiting enzyme in melatonin synthesis (7-9). Additionaly, cyclic AMP controls the expression of immediate early genes capable of further modulating cyclic AMP-responsive element (CRE) responsiveness (10-12). In particular, the mRNA of inducible cyclic AMP early repressor (ICER), a known cyclic AMP-inducible repressor of cyclic AMP-dependent transcription (13,14), obeys a dramatic circadian pattern of expression, suggesting the existence of a feedback control between CREB activation and CRE-responsive gene transcription (11,12).NE increases the amount of cyclic AMP through both ,3-and al-noradrener...
SUMMARY:The aim of the study was to assess the sensitivity and specificity of fluorescence immunocytochemistry (uCytϩ assay) as combined with urinary cytology for detection of primary and recurrent urothelial carcinomas. We analyzed 694 urinary samples from 236 new symptomatic patients and 458 patients followed after transurethral resection (TUR) for bladder tumor. Lesions suspicious for cancer at cystoscopy were sampled by biopsies or TUR. Sensitivity and specificity of tests were calculated using cystoscopy and histopathology, whether or not combined as gold standards. In new symptomatic patients, sensitivity of uCytϩ was 40%, 88.2%, and 76.7%, whereas that of urinary cytology was 30%, 70.6%, and 83.3%, respectively, in G1, G2, and G3 tumors. In follow-up cases, sensitivity of uCytϩ was 61.9%, 66.7%, and 76.9%, whereas that of urinary cytology was 38.1%, 58.3%, and 64.1%, respectively, in G1, G2, and G3 tumors. The combination of uCytϩ and urinary cytology significantly increased mean sensitivity in newly diagnosed cases (86.4% versus 71.2% with urinary cytology only, p Ͻ 0.05), as well as in patients followed after TUR (79.3% versus 55.2%, p Ͻ 0.001). Specificity of uCytϩ and urinary cytology was identical in new patients (83.3%) and was 81.9% and 86.2%, respectively, in patients followed after TUR. In patients with negative cystoscopy, positive uCytϩ tests had a strong predictive value for tumor recurrence at 1 year (47.0% versus 11.9% in patients with negative assay, p Ͻ 0.01). We conclude that combining uCytϩ with urinary cytology improves the detection of urothelial carcinomas as well in patients with symptoms suggesting bladder cancer as in those followed after treatment. (Lab Invest 2003, 83:845-852).
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