Clinical and genetic study of 46 Italian patients with primary lymphedema.

Primary lymphedema is characterized by altered morphological development of lymphatic vessels causing fluid accumulation in interstitial spaces. In familial forms, it is primarily transmitted as a dominant Mendelian trait with heterozygous mutations in genes involved in lymphangiogenesis. We used PCR and direct sequencing to analyze the region of the fms-related tyrosine kinase 4 (FLT4) gene encoding the "tyrosine-kinase domain" and the single exon of the forkhead box C2 (FOXC2) gene in 46 Italian probands with primary lymphedema, 42 of whom had familial forms. We identified 12 mutations in 12 patients (12/46, 26%), six in the FLT4 gene and six in the FOXC2 gene. Most of the mutations (9/12, 75%) were new, and none were identified in 100 healthy subjects or listed in the NCBI dbSNP. A clear relation emerged between genotype and phenotype because 4/5 (80%) probands with onset at birth showed FLT4 mutations and 4/5 (80%) probands without distichiasis and with FOXC2 mutations had an amino-acid substitution outside the forkhead domain. Besides the allelic heterogeneity shown by unique mutations in each proband, the absence of mutations in almost 75% of familial cases of primary lymphedema also suggests genetic heterogeneity.

Memory-Efficient Training for Fully Unrolled Deep Learned PET Image Reconstruction with Iteration-Dependent Targets.

We propose a new version of the forward-backward splitting expectation-maximisation network (FBSEM-Net) along with a new memory-efficient training method enabling the training of fully unrolled implementations of 3D FBSEM-Net. FBSEM-Net unfolds the maximum a posteriori expectation-maximisation algorithm and replaces the regularisation step by a residual convolutional neural network. Both the gradient of the prior and the regularisation strength are learned from training data. In this new implementation, three modifications of the original framework are included. First, iteration-dependent networks are used to have a customised regularisation at each iteration. Second, iteration-dependent targets and losses are introduced so that the regularised reconstruction matches the reconstruction of noise-free data at every iteration. Third, sequential training is performed, making training of large unrolled networks far more memory efficient and feasible. Since sequential training permits unrolling a high number of iterations, there is no need for artificial use of the regularisation step as a leapfrogging acceleration. The results obtained on 2D and 3D simulated data show that FBSEM-Net using iteration-dependent targets and losses improves the consistency in the optimisation of the network parameters over different training runs. We also found that using iteration-dependent targets increases the generalisation capabilities of the network. Furthermore, unrolled networks using iteration-dependent regularisation allowed a slight reduction in reconstruction error compared to using a fixed regularisation network at each iteration. Finally, we demonstrate that sequential training successfully addresses potentially serious memory issues during the training of deep unrolled networks. In particular, it enables the training of 3D fully unrolled FBSEM-Net, not previously feasible, by reducing the memory usage by up to 98% compared to a conventional end-to-end training. We also note that the truncation of the backpropagation (due to sequential training) does not notably impact the network's performance compared to conventional training with a full backpropagation through the entire network.

ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex.

The small GTPase ADP-ribosylation factor (ARF) regulates the structure and function of the Golgi complex through mechanisms that are understood only in part, and which include an ability to control the assembly of coat complexes and phospholipase D (PLD). Here we describe a new property of ARF, the ability to recruit phosphatidylinositol-4-OH kinase-beta and a still unidentified phosphatidylinositol-4-phosphate-5-OH kinase to the Golgi complex, resulting in a potent stimulation of synthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate; this ability is independent of its activities on coat proteins and PLD. Phosphatidylinositol-4-OH kinase-beta is required for the structural integrity of the Golgi complex: transfection of a dominant-negative mutant of the kinase markedly alters the organization of the organelle.

Role of NAD+ and ADP-ribosylation in the maintenance of the Golgi structure.

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114-1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065-7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre-ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200-14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre-ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50-enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

How fish power swimming.

It is thought that fish generate the power needed for steady swimming with their anterior musculature, whereas the posterior musculature only transmits forces to the tail and does negative work. Isolated red muscle bundles driven through the length changes and stimulation pattern that muscles normally undergo during steady swimming showed the opposite pattern. Most of the power for swimming came from muscle in the posterior region of the fish, and relatively little came from the anterior musculature. In addition, the contractile properties of the muscle along the length of the fish are significantly adapted to enhance power generation.

Fast receptor-induced formation of glycerophosphoinositol-4-phosphate, a putative novel intracellular messenger in the Ras pathway.

Glycerophosphoinositols are phosphoinositide metabolites whose levels are constitutively elevated in Ras-transformed cells. Here, we show that one of these compounds, glycerophosphoinositol-4-phosphate (GroPIns-4-P) responds acutely to the stimulation of the epidermal growth factor receptor, with a fast, massive and transient increase. The mechanism leading to GroPIns-4-P formation involves the activation of phosphoinositide-3 kinase and the small GTP-binding protein Rac, since GroPIns-4-P was neither formed in cells expressing the dominant negative form of Rac nor in cells treated with the phosphoinositide-3 kinase inhibitor wortmannin. GroPIns-4-P has been previously shown to inhibit adenylyl cyclase. Accordingly, epidermal growth factor also decreased the basal, cholera toxin-stimulated, and forskolin-stimulated cyclic AMP levels with kinetics similar to those of GroPIns-4-P formation, suggesting that GroPIns-4-P mediates this inhibitory effect. The hormone-induced formation of GroPIns-4-P was detected in several cell lines of various origin, suggesting that GroPIns-4-P is a novel intracellular messenger of the Ras pathway, possibly able to convey information from tyrosine kinase receptors to the cyclic AMP cascade.

Release of the mitogen lysophosphatidylinositol from H-Ras-transformed fibroblasts; a possible mechanism of autocrine control of cell proliferation.

Lysophosphatidylinositol (LysoPtdIns) is formed by a constitutively-active phosphoinositide-specific phospholipase A2 in Ras-transformed cells and can stimulate cell proliferation. To evaluate whether LysoPtdIns could function as an autocrine modulator of cell growth, we examined whether LysoPtdIns can be released in the medium of Ras-transformed FRT-Fibro fibroblasts and thyroid cells. Here, we report that LysoPtdIns accumulates in the extracellular space of these lines and reaches levels up to tenfold higher than in the case of normal cells. Moreover, the ionophore A23187 increased the levels of the lysolipid in the extracellular medium. Extracellular LysoPtdIns was rapidly hydrolyzed to inositol 1:2-cyclic phosphate. LysoPtdIns induced thymidine incorporation in FRT-Fibro Ha-Ras fibroblasts, whereas inositol cyclic 1:2-cyclic phosphate did not affect cell growth per se, nor did it interfere with the LysoPtdIns mitogenic activity. We hypothesize that in Ras-transformed fibroblasts the formation and release of LysoPtdIns may function as an autocrine mechanism that participates in the Ras-dependent stimulation of cell growth.

Signalling pathways involved in the mitogenic action of lysophosphatidylinositol.

Lysophosphatidylinositol has been previously shown to stimulate cell proliferation in differentiated and in K-ras transformed thyroid cells. Increased levels of lysophosphatidylinositol, but not lysophosphatidylcholine or lysophosphatidylethanolamine, are present in thyroid as well as in other ras-transformed cell lines. We have now investigated the mechanism of action of this lysolipid by analysing its effects in a differentiated thyroid cell line. Lysophosphatidylinositol did not increase the levels of cAMP, the main stimulator of cell proliferation in the thyroid, whereas it stimulated phosphoinositide breakdown, mobilization of cytosolic Ca2+ and arachidonic acid release, suggesting that it activates both phospholipases C and A2. None of the effects of lysophosphatidylinositol were prevented by pretreatment of cells with pertussis toxin. Instead, the tyrosine kinase inhibitors, tyrphostins AG18 and AG561, completely blocked its mitogenic action. The effects of lysophosphatidylinositol were distinguishable from those of the well known mitogen lysophosphatidic acid, which affected differently the signalling pathways analysed and was not mitogenic in ras-transformed cells. These results suggest that the mitogenic activity of lysophosphatidylinositol is associated with the activation of phospholipase C and phospholipase A2 and is relatively specific for ras-transformed cells.

Two novel mutations of the AIRE protein affecting its homodimerization properties.

We report two novel mutations, c.230T>C (p.F77S) and c.64_69del (p.V22_D23del) within the HSR domain of the AIRE protein in two patients of Italian descent affected by APECED. Both mutations were found in the compound heterozygous state respectively with c.994+5G>T and c.232T>A (p.W78R). With the two-hybrid assay in the yeast system we found that constructs containing the two mutations fail to interact with the wild-type protein. These findings indicate that both mutations negatively affected the homodimerization properties of the AIRE protein, thereby leading to a defective function.

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype.

Phorbol myristate acetate inhibits alpha 1-adrenergically but not thyrotropin-regulated functions in FRTL-5 rat thyroid cells.

The iodination of thyroglobulin and the formation of thyroid hormones are regulated by alpha 1-adrenergic agents as well as TSH in rat FRTL-5 cells. The regulatory effects of the alpha-1-adrenergic agents and TSH on both of these processes are associated with an increase in cytosolic Ca2+ and an increase in that component of iodide efflux that is representative of the movement of iodide from the thyroid cell into the follicular lumen. When FRTL-5 cells are preincubated with phorbol myristate acetate (PMA) for at least 3 min, the norepinephrine-stimulated changes in cytosolic Ca2+ levels and iodide efflux are inhibited. In contrast, PMA pretreatment has no effect on iodide efflux and actually enhances the changes in cytosolic Ca2+ induced by TSH. Phorbol myristate acetate pretreatment has no effect on TSH-stimulated cAMP-mediated iodide uptake in FRTL-5 cells, nor does it affect the binding parameters of the alpha 1-adrenergic receptor antagonist prazosin. These data suggest that protein kinase C is involved in a feedback mechanism regulating alpha 1-adrenergic but not TSH-induced changes associated with the iodination of thyroglobulin and the formation of thyroid hormones; and that this feedback effect occurs after the step of ligand binding but before the increase in cytosolic Ca2+ induced by the alpha 1-adrenergic agents.

Immunoglobulins from Graves' patients stimulate phospholipase-A2 in FRTL5 thyroid cells.

The well documented ability of immunoglobulins G (IgGs) from Graves' patients to stimulate cAMP production is believed to be involved in the pathophysiology of this disease. It is still under discussion whether other intracellular messengers known to regulate thyroid function might play a similar role. This study shows that phospholipase-A2, a signal pathway unrelated to cAMP, is activated by Graves' IgGs. The IgGs from 67 patients with active Graves' disease, 8 patients with Graves' disease in remission, 5 patients with idiopathic myxedema, 2 patients with Hashimoto's thyroiditis, 57 patients with nonautoimmune thyroid disease, and 65 normal subjects were tested for their ability to stimulate phospholipase-A2 activity, as measured by arachidonic acid release from FRTL5 thyroid cells. The IgGs from patients with active Graves' disease caused a significant increase in arachidonic acid release compared to those from normal subjects, patients with nonautoimmune thyroid diseases, and patients with Graves' disease in remission (P less than 0.0001). The IgGs from active Graves' patients were also able to increase cAMP accumulation in FRTL5 cells. This effect did not correlate with the ability of the same IgGs to induce arachidonic acid release, suggesting that Graves' IgGs stimulate these two pathways by separate mechanisms. Moreover, a subgroup of IgGs that stimulated phospholipase-A2 did not increase the cAMP levels in FRTL5 cells. Our data suggest a novel mechanism of action of Graves' IgGs, the activation of phospholipase-A2, well distinguishable from the known effect on cAMP accumulation. The assay we describe could be helpful in improving the diagnosis and therapy of Graves' disease and in distinguishing it from nonautoimmune thyroid diseases. It also supplies the basis for a prospective subclassification of the Graves' patients, which might become useful to clarify the pathophysiology of this disease.

Cyclooxygenase-dependent thyroid cell proliferation induced by immunoglobulins from patients with Graves' disease.

IgG associated with Graves' disease bind to the TSH receptor and alter thyroid growth and function, mainly through the stimulation of adenylyl cyclase. In addition, Graves' IgG are able to interact with the phospholipase C (PLC)/Ca2+ and phospholipase A2 (PLA2)/arachidonic acid (AA) cascades. The activation of this latter pathway leads to thyroid cell growth in vitro. The elucidation of additional mechanisms of action of Graves' IgG has made possible the identification of four subgroups of patients, characterized by IgG with different biochemical activities (extent of cAMP and AA release stimulation in in vitro assays). On the basis of these results, a novel therapeutic approach could be proposed based on the inhibition of PLA2 and AA metabolism. To test this hypothesis, the ability of IgG from 56 Graves' patients to stimulate [3H]thymidine incorporation in FRTL5 thyroid cells in the presence and absence of the cyclooxygenase inhibitor indomethacin (2.5 x 10(-6) mol/L) was measured. A significant reduction in [3H]thymidine incorporation was found (33% inhibition; P < 0.0001) upon pretreatment with indomethacin, suggesting that in vitro thyroid cell growth is regulated by cyclooxygenase metabolites. This strengthens the argument for involvement of the PLA2/AA cascade in the pathophysiology of Graves' disease and the proposal for novel selective pharmacological treatments of these patients.

Subgroups of Graves' patients identified on the basis of the biochemical activities of their immunoglobulins.

Graves' patient immunoglobulins (IgG) are known to activate adenylyl cyclase. Recently, we have shown that they also stimulate phospholipase A2 (PLA2). Here we analyze the relationship of these biochemical activities of Graves' IgG to thyroid growth in vitro ([3H]thymidine incorporation) and in vivo (patient goiter size) as well as to clinical indicators of severity of the disease, such as ophthalmopathy, T3 levels, T3/T4 molar ratio, and TSH binding-inhibiting IgG activity. A cluster analysis of the biochemical parameters referring to the whole population (158 subjects) led to the identification of 4 subgroups of Graves' patients based on the different capabilities of IgG to stimulate adenylyl cyclase, PLA2, and [3H]thymidine incorporation. Importantly, a trend of increasing severity of the disease from group 1 to group 4 could be identified. In particular, patients in group 4 (characterized by elevated stimulation of adenylyl cyclase, PLA2, and [3H]thymidine incorporation) had the largest goiter, highest serum concentration of T3, highest T3/T4 molar ratio, and highest prevalence of ophthalmopathy. These results indicate that Graves' IgG induce thyroid growth by stimulating both adenylyl cyclase and PLA2, and suggest a method for the subclassification of Graves' patients that identifies four groups with different degrees of severity of the disease. Moreover, this classification might lead to the targeted use of a novel therapeutic approach based on the inhibition of PLA2 and arachidonic acid metabolism.

Physiological concentrations of thyrotropin increase cytosolic calcium levels in primary cultures of human thyroid cells.

The activity of TSH, the main regulator of growth and differentiation in the thyroid, has been mainly related to the activation of the adenylyl cyclase cascade. TSH also activates phospholipase-C and -A2; these effects, however, have been reported to require concentrations of the hormone up to 1000-fold higher than those effective on adenylyl cyclase, suggesting that the main physiological mechanism involved in the action of TSH is the activation of this enzyme. Using primary cultures of human thyroids, we here show that physiological concentrations of TSH (0.01-10 mU/L) are also able to increase intracellular Ca2+ levels. Cells were loaded with the fluorescent Ca2+ probe fura-2 and analyzed by single cell Ca2+ recording. The basal Ca2+ level was 105 +/- 30 nmol/L, and physiological concentrations of TSH increased it by 2- to 7-fold. The Ca2+ increase was transient and lasted up to 10 min. It is also shown that the TSH-dependent Ca2+ increase involves both the activation of phospholipase-C and the entry of extracellular Ca2+. TSH (100-10000 mU/L) increased cAMP levels by up to 20-fold in parallel experiments performed on the same cell preparations. These data demonstrate that physiological concentrations of TSH are able to increase cytosolic Ca2+ levels, indicating that this second messenger might directly mediate the action of this hormone in the thyroid.

Graves' immunoglobulins activate phospholipase A2 by recognizing specific epitopes on thyrotropin receptor.

Thyroid-stimulating IgG from Graves' patients bind to the TSH receptor and activate both adenylyl cyclase (AC) and phospholipase A2 (PLA2) in FRTL5 thyroid cells. Both activities have been associated with increased thyroid cell growth and function; evidence exists that subpopulations of Graves' IgG can stimulate either AC or PLA2 cascades and that the activation of both is associated with the largest goiters in patients. Studies using chimeras of the human TSHR receptor (hTSHR) and the LH-CG receptor show that most patients with Graves' disease have cAMP-stimulating IgG that require epitopes on the N-terminal portion of the TSHR extracellular domain; epitopes associated with PLA2 activation are not clear. To address this question we used stably transfected Chinese hamster ovary (CHO) cells containing the wild-type hTSHR and the hTSHR chimera with residues 8-165 (Mc1+2) substituted by equivalent residues of the LH-CG receptor. PLA2 activity, measured as arachidonic acid (AA) release, was determined in 32 patients with Graves' disease. We show that 72% of Graves' patients have IgG able to stimulate PLA2 in CHO cells transfected with the TSHR and that AA release induced by Graves' IgG was significantly reduced (P = 0.022) in the CHO-Mc1+2-transfected cells (193 +/- 88% vs. 131 +/- 67%, respectively). Unlike IgG, the effect of TSH was not modified in the CHO-Mc1+2-transfected cells. When we compared the AC- and PLA2-stimulating activities of these 32 IgG in wild-type TSHR transfectants, we found that 63% of Graves' patients have antibodies able to stimulate both PLA2 and AC, whereas some patients' IgG were active only in AC or PLA2 assays. Of the patients with IgG having activity in both assays in wild-type TSHR transfectants, 50% of the IgG lost their stimulatory activities in both AA release and cAMP assays in Mc1+2 cells. Of the remainder, some IgG maintained their activity in one (AA release) or the other (cAMP) assay when measured in Mc1+2 chimeras. Thus, our data show that the N-terminal portion of extracellular domain of the TSHR is required for PLA2 as well as AC activation by IgG from patients with Graves' disease. These data also demonstrate that patients with Graves' disease have heterogeneous autoantibodies that selectively activate AC and PLA2 pathways and suggest that patients with autoantibodies active in both assays have more severe disease, with higher thyroid hormone levels and larger goiters.

Adenylate cyclase activity of v-ras-k transformed rat epithelial thyroid cells.

The regulation of adenylate cyclase has been analyzed in normal rat thyroid cells as well as in the same cells transformed by the v-ras-k oncogene. In both cell types the adenylate cyclase complex consists of the two GTP-binding proteins, Gi and Gs, as demonstrated by the specific ADP-ribosylation induced by pertussis and cholera toxin, respectively. The response of adenylate cyclase of the transformed cells to forskolin, pertussis toxin and cholera toxin is attenuated with respect to the control cell line. The thyrotropic hormone (TSH), that acts on normal thyroid cells in culture as a growth factor by stimulating the adenylate cyclase activity, is not able to induce DNA synthesis nor does it stimulate adenylate cyclase in v-ras-k transformed cells.

PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes.

We reported that an inhibitor of sphingolipid biosynthesis, D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al., 1998, J. Cell Biol. 142, 25-38). We now show that PDMP partially blocks the BFA-induced ADP-ribosylation of the cytosolic protein BARS-50. Moreover, PDMP does not interfere with the BFA-induced inhibition of the binding of ADP-ribosylation factor (ARF) and the coatomer component beta-coat protein to Golgi membranes. These results are consistent with a role of ADP-ribosylation in the action of BFA and with the involvement of BARS-50 in the regulation of membrane trafficking.

Maintenance of PtdIns45P2 pools under limiting inositol conditions, as assessed by liquid chromatography-tandem mass spectrometry and PtdIns45P2 mass evaluation in Ras-transformed cells.

Inositol-containing molecules are involved in important cellular functions, including signalling, membrane transport and secretion. Our interest is in lysophosphatidylinositol and the glycerophosphoinositols, which modulate cell proliferation and G-protein-dependent activities such as adenylyl cyclase and phospholipase A(2). To investigate the role of glycerophosphoinositol (GroPIns) in the modulation of Ras-dependent pathways and its correlation to Ras transformation, we employed a novel liquid chromatography-tandem mass spectrometry technique to directly measure GroPIns in cell extracts. The cellular levels of GroPIns in selected parental and Ras-transformed cells, and in some carcinoma cells, ranged from 44 to 925 microM, with no consistent correlation to Ras transformation across all cell lines. Moreover, the derived cellular inositol concentrations revealed a wide range ( approximately 150 microM to approximately 100 mM) under standard [(3)H]-inositol-loading, suggesting a complex relationship between the inositol pool and the phosphoinositides and their derivatives. We have investigated these pools under specific loading conditions, designing a further HPLC analysis for GroPIns, combined with mass determinations of cellular phosphatidylinositol 4,5-bisphosphate. The data demonstrate that limiting inositol conditions identify a preferred pathway of inositol incorporation and retention into the polyphosphoinositides pool. Thus, under conditions of increased metabolic activity, such as receptor stimulation or cellular transformation, the polyphosphoinositide levels will be maintained at the expense of phosphatidylinositol and the turnover of its aqueous derivatives.

Signaling pathways involved in thyroid hyperfunction and growth in Graves' disease.

The elucidation of the multiple signaling cascades coupled to the TSH receptor has offered new approaches in the understanding of the pathogenesis of Graves' disease. Here we review findings showing that immunoglobulins from Graves' patients are heterogeneous, bind to different epitopes and, similarly to TSH, activate different signaling pathways, including adenylyl cyclase, phospholipase C and phospholipase A2. Evidence that the multiplicity of signals correlates with the different manifestations of the disease is also summarized. We believe that the dissection of the molecular mechanisms involved in the pathogenesis of Graves' disease offers the basis for developing novel therapeutical approaches to this disease.