A new case of Carney triad: gastrointestinal stromal tumours and leiomyoma of the oesophagus do not show activating mutations of KIT and platelet-derived growth factor receptor alpha.

The Carney triad is a rare syndrome of unknown aetiology, with synchronous or metachronous appearance of rare neoplasms: gastrointestinal stromal tumours (GISTs), pulmonary chondromas and extra-adrenal paragangliomas. In most cases, the Carney triad is incomplete. The combination encountered typically, GISTs and pulmonary chondromas, was also seen in our patient, a 22-year-old woman. She was diagnosed with the triad after Billroth II gastrectomy for histologically proved gastric GISTs. The diagnosis of pulmonary chondromas was confirmed by transthoracic, computed tomography-guided needle biopsy. An oesophageal leiomyoma was resected 2 years after the initial diagnosis, on suspicion of paraganglioma. The clinical course of the patient has been uneventful since. The last follow-up was carried out 6 years after the initial diagnosis. On histological examination, the cells of gastric GIST were partly positive for CD34, whereas CD117 was expressed in all areas in variable intensity and S-100 protein was negative. The oesophageal tumour was classified as leiomyoma due to strong immunopositivity for smooth muscle actin and desmin, being negative for CD34 and CD117. Two different gastric GIST lesions as well as the oesophageal leiomyoma and normal tissue were analysed for activating mutations in common hot spots of KIT (exon 9 and 11) and platelet-derived growth factor receptor alpha (exon 18), but in all probes wild-type sequences were found. These results are in accordance with the first published analyses of GIST lesions from Carney patients.

Analyses of signal transduction cascades reveal an essential role of calcium ions for regulation of melatonin biosynthesis in the light-sensitive pineal organ of the rainbow trout (Oncorhynchus mykiss).

Signal transduction processes regulating melatonin production in the light-sensitive trout pineal organ were investigated by immunocytochemical and immunochemical demonstration of phosphorylated cyclic AMP-responsive element-binding protein (pCREB) and measurements of cyclic AMP, melatonin, and calcium levels. Melatonin levels were tightly controlled by light and darkness. Elevation of cyclic AMP levels by 8-bromo-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine increased the levels of pCREB and melatonin in light- or dark-adapted pineal organs in vitro. Without pharmacological treatment, the levels of pCREB and cyclic AMP remained constant for several hours before and after light onset. Inhibition of cyclic AMP-dependent proteasomal proteolysis by lactacystin, MG 132, and calpain inhibitor I did not prevent the rapid, light-induced suppression of melatonin biosynthesis. However, changes in the intracellular calcium concentration by drugs affecting voltage-gated calcium channels of the L type and intracellular calcium oscillations (cobalt chloride, nifedipine, Bay K 8644) had dramatic effects on the rapid, light-dependent changes in melatonin levels. These effects were not accompanied by changes in cyclic AMP levels. Thus, the rapid, light-dependent changes in melatonin levels in the trout pineal organ are regulated apparently by a novel calcium signaling pathway and do not involve changes in cyclic AMP levels, cyclic AMP-dependent proteasomal proteolysis, or phosphorylation of cyclic AMP-responsive element-binding protein.

Signal transduction in the rodent pineal organ. From the membrane to the nucleus.

The rodent pineal organ transduces a photoneural input into a hormonal output. This photoneuroendocrine transduction leads to highly elevated levels of the hormone melatonin at night-time which serves as a message for darkness. The melatonin rhythm depends on transcriptional, translational and posttranslational regulation of the arylalkylamine-N-acetyltransferase, the key enzyme of melatonin biosynthesis. These regulatory mechanisms are fundamentally linked to two second messenger systems, namely the cAMP- and the Ca(2+)-signal transduction pathways. Our data gained by molecular biology, immunohistochemistry and single-cell imaging demonstrate a time- and substance-specific activation of these signaling pathways and provide a framework for the understanding of the complex signal transduction cascades in the rodent pineal gland which in concert not only regulate the basic profile but also fine-tune the circadian rhythm in melatonin synthesis.

Lymph node micrometastases of cutaneous melanoma: increased sensitivity of molecular diagnosis in comparison to immunohistochemistry.

The presence of regional lymph node metastases is one of the most significant prognostic factors for predicting survival in patients with clinical stage I or II cutaneous melanoma. For accurate staging of the primary tumor a sensitive technique is required to detect occult nodal micrometastases. This prospective diagnostic study was designed to evaluate the incidence of nodal micrometastases using nested reverse transcription-polymerase chain reaction (RT-PCR) for tyrosinase in comparison to immunohistochemical examination. Furthermore, the incidence of melanoma micrometastases detected by RT-PCR was analysed in correlation to major prognostic factors. A total of 466 regional lymph nodes from 79 patients with primary cutaneous melanoma (tumor thickness > 0.75 mm) were investigated. In 49 lymph nodes from 31 patients immunohistochemistry demonstrated melanoma metastases. Using tyrosinase RT-PCR, nodal micrometastases were detected in 136 lymph nodes from 52 patients including all lymph nodes positive by immunohistochemical examination. Out of the 417 lymph nodes negative by immunohistochemistry, 87 nodes (21%) were identified to express tyrosinase by the RT-PCR technique. Among the 48 patients negative by immunohistochemical assessment, 21 (44%) had nodal micrometastases (n = 40) using RT-PCR. All 68 lymph nodes from 46 non-melanoma patients serving as negative controls for tyrosinase RT-PCR were negative. The detection of melanocytic nodal micrometastases by tyrosinase RT-PCR is a highly specific method with a sensitivity significantly higher than that achieved by immunohistochemistry (p < 0.0001). Patients with nodal micrometastases identified exclusively by RT-PCR had significantly higher tumor thickness as compared to patients with negative results by RT-PCR (p < 0.01).

A specific and sensitive double-immunofluorescence method for the demonstration of S-antigen and serotonin in trout and rat pinealocytes by means of primary antibodies from the same donor species.

Immunocytochemical double-labeling methods are important tools in cell and neurobiology. Here we describe a method which is based on double immunofluorescence and allows specific detection of two different antigens located in the same cell compartment by two primary antibodies raised in the same species. As an example, we present the double-immunolabeling method for the S-antigen (SAg), a photoreceptor-specific protein, and the indoleamine serotonin (5HT) in dissociated trout and rat pineal cells immobilized on coverslipped and in frozen sections of the trout pineal organ. As a first step, the preparations on the slides or coverslips were sequentially incubated with the first primary antibody (rabbit anti-SAg), the fluorescein-labeled (anti-rabbit) secondary antibody, and then with normal rabbit serum. Meanwhile, the second primary antibody (rabbit anti-5HT) was coupled to a Cy3-labeled secondary (anti-rabbit) antibody in a reaction tube and excess binding sites were quenched with normal rabbit serum. This complex was applied to the specimens after completion of the first (SAg) immunoreaction on the slide. For control experiments, the first (anti-SAg) or the second (anti-5HT) primary antibody were omitted. Most of the rat and trout pinealocytes were double immunolabeled for SAg and 5HT. In the trout, few cells contained SAg or 5HT immunoreaction only. This underlines the selectivity of each immunoreaction. The results show that the method can be used for the analysis of whole cells and tissue sections by means of conventional fluorescence and confocal laser scanning microscopy.

Regulation of the intracellular concentration of free calcium ions in pinealocytes of the rainbow trout and the rat.

Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout pinealocytes and in rat pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout pinealocyte. In rat pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in pinealocytes are complex and differ considerably between poikilothermic and mammalian species.

Calcium oscillations in a subpopulation of S-antigen-immunoreactive pinealocytes of the rainbow trout (Oncorhynchus mykiss).

By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca2+]i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca2+]i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Application of 60 mM KCl elevated [Ca2+]i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 microM nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca2+]i remains elusive. Treatment with norepinephrine (100 pM-50 microM), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca2+]i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.

Regulation of melatonin production and intracellular calcium concentrations in the trout pineal organ.

The present in vitro study correlates measurements of the melatonin production from trout pineal organs with those of the intracellular calcium concentration in pinealocytes. Melatonin production increases with decreasing irradiance and shows maximal values in darkness. Some pinealocytes exhibit spontaneous calcium oscillations, although most of them have a stable basal calcium concentration. Diminishing extracellular calcium and enhancing magnesium reduces melatonin release in the light-and dark-adapted state. The application of Co2+ decreases melatonin secretion in the mesopic and scotopic range, reversibly blocks spontaneous calcium oscillations, reduces the basal intracellular calcium concentration in non-oscillating pinealocytes, and inhibits the KCl-induced rise in intracellular calcium. Application of glutamate, norepinephrine, isoproterenol, or dopamine has no significant effect on melatonin secretion. Norepinephrine does not influence the calcium concentration in any of the trout pinealocytes. Treatment with the GABAA-receptor agonist muscimol causes a slight reduction of melatonin release in the mesopic and scotopic range of illumination, without affecting intracellular calcium concentrations. Thus, Co2+ and low calcium/high magnesium buffer reduce melatonin release through an action on the calcium concentration in trout pinealocytes and not through a blockade of synaptic transmission. All the data show that the trout pineal organ synthesizes and releases melatonin in relation to the irradiance of the incident light and that neuronal inputs have a minor, if any, influence on melatonin synthesis.