Conformation dependent pro-apoptotic activity of the recombinant human prion protein fragment 90-231.

The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in alpha and beta conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53 degrees C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high beta sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native alpha-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a beta-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.

Identification of a conserved N-capping box important for the structural autonomy of the prion alpha 3-helix: the disease associated D202N mutation destabilizes the helical conformation.

Peptides corresponding to three alpha helices present in the C-terminal region of the human prion protein have been synthesized and their structural autonomy analyzed by circular dichroism (CD) and NMR spectroscopy. The results obtained indicate that the protein fragment corresponding to the alpha 3-helix, in contrast to alpha 1 and alpha 2 peptides, shows a complete structural autonomy. The chemical shifts values found for NH and CHalpha resonance of the isolated alpha 3 peptide, formed by 30 aminoacid residues, were markedly and surprisingly similar to the corresponding values of the alpha 3-helix in the protein. The structural autonomy of the alpha 3-helix is profoundly determined by the presence of the conserved capping box and, in part, by the ionic bond formed between Glu200 and Lys204. On the basis of these observations a novel PrP consensus pattern, centered on the alpha 3-helix region, has been defined. The data indicate that this autonomous and highly conserved region of the PrPc likely plays a critical role in folding and stability. This gives an explanation of why many of pathogenic mutations occur in this part of the molecule, sharing relevant effects on the overall protein conformation. In particular the D202N capping mutation almost completely destabilizes the isolated alpha 3 peptide. While it is well known that the D202N substitution is associated with a GSS disease, the possible structural basis of this fatal pathology has never been investigated. We propose that a lower alpha 3-helical propensity leading to a major destabilization of the PrPc molecule initiates the pathogenic process associated with D202N capping mutation.

The activation of the phosphotyrosine phosphatase eta (r-PTP eta) is responsible for the somatostatin inhibition of PC Cl3 thyroid cell proliferation.

The aim of this study was the characterization of the intracellular effectors of the antiproliferative activity of somatostatin in PC Cl3 thyroid cells. Somatostatin inhibited PC Cl3 cell proliferation through the activation of a membrane phosphotyrosine phosphatase. Conversely, PC Cl3 cells stably expressing the v-mos oncogene (PC mos) were completely insensitive to the somatostatin antiproliferative effects since somatostatin was unable to stimulate a phosphotyrosine phosphatase activity. In PC mos cells basal phosphotyrosine phosphatase activity was also reduced, suggesting that the expression of a specific phosphotyrosine phosphatase was impaired in these transformed cells. We suggested that this phosphotyrosine phosphatase could be r-PTP eta whose expression was abolished in the PC mos cells. To directly prove the involvement of r-PTP eta in somatostatin's effect, we stably transfected this phosphatase in PC mos cells. This new cell line (PC mos/PTP eta) recovered somatostatin's ability to inhibit cell proliferation, showing dose-dependence and time course similar to those observed in PC Cl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTP eta did not restore the antiproliferative effects of somatostatin. PC mos/PTP eta cells showed a high basal phosphotyrosine phosphatase activity which, similarly to PC Cl3 cells, was further increased after somatostatin treatment. The specificity of the role of r-PTP eta in somatostatin receptor signal transduction was demonstrated by measuring its specific activity after somatostatin treatment in an immunocomplex assay. Somatostatin highly increased r-PTP eta activity in PCCl3 and PC mos/PTP eta (+300%, P < 0.01) but not in PCmos cells. Conversely, no differences in somatostatin-stimulated SHP-2 activity, (approximately +50%, P < 0.05), were observed among all the cell lines. The activation of r-PTP eta by somatostatin caused, acting downstream of MAPK kinase, an inhibition of insulin-induced ERK1/2 activation with the subsequent blockade of the phosphorylation, ubiquitination, and proteasome degradation of the cyclin-dependent kinase inhibitor p27(kip1). Ultimately, high levels of p27(kip1) lead to cell proliferation arrest. In conclusion, somatostatin inhibition of PC Cl3 cell proliferation requires the activation of r-PTP eta which, through the inhibition of MAPK activity, causes the stabilization of the cell cycle inhibitor p27(kip1).

Oncogene transformation of PC Cl3 clonal thyroid cell line induces an autonomous pattern of proliferation that correlates with a loss of basal and stimulated phosphotyrosine phosphatase activity.

The effects of the stable expression of E1A and/or middle T oncogenes on the proliferative activity of PC Cl3 normal thyroid cells are reported. The proliferation of PC Cl3 cells is mainly regulated by insulin and TSH in a stimulatory way and by somatostatin in an inhibitory fashion. The transformed cell lines, named PC Py and PC E1A Py, show an autonomous pattern of proliferation. The blockade of phosphotyrosine phosphatase activity with vanadate increased the proliferation rate of PC Cl3 under basal and stimulated conditions and completely prevented the inhibitory activity of somatostatin, suggesting that in PC Cl3 cells, a tonic tyrosine phosphatase activity regulates basal and stimulated proliferation, and that a somatostatin-dependent increase in this activity may represent a cytostatic signal. Conversely, in both PC Py and PC E1A Py, vanadate did not modify basal and stimulated proliferation. We analyzed tyrosine phosphatase activity in the different cell lines basally and under conditions leading to the arrest of cell proliferation: confluence (contact inhibition), growth factor deprivation (starvation), and somatostatin treatment. Under basal conditions, tyrosine phosphatase activity was significantly lower in PC Py and PC E1APy cell lines than that in the normal cells. The inhibition of the proliferation induced by contact inhibition or somatostatin treatment was accompanied by an increase in tyrosine phosphatase activity only in PC Cl3 cells. The reduction in tyrosine phosphatase activity in PC E1APy cells correlated with a significant reduction in the expression of R-PTP eta, a tyrosine phosphatase cloned from PC Cl3 cells. Conversely, the expression of another receptor-like PTP, PTP mu, was unchanged. Thus, PTP eta may be a candidate to mediate inhibitory signals (i.e. activation of somatostatin receptors or cell to cell contact) on the proliferative activity of PC Cl3 cells, and the reduction of its expression in the transformed cell lines may lead to an alteration in the control of cell proliferation.

Somatostatin inhibits tumor angiogenesis and growth via somatostatin receptor-3-mediated regulation of endothelial nitric oxide synthase and mitogen-activated protein kinase activities.

Somatostatin was reported to inhibit Kaposi's sarcoma (KS) cell (KS-Imm) xenografts through an antiangiogenic activity. Here, we show that somatostatin blocks growth of established KS-Imm tumors with the same efficacy as adriamycin, a clinically effective cytotoxic drug. Whereas KS-Imm cells do not express somatostatin receptors (SSTRs), endothelial cells express several SSTRs, in particular SSTR3. We investigated the molecular mechanisms and receptor specificity of somatostatin inhibition of angiogenesis. Somatostatin significantly inhibited angiogenesis in vivo in the matrigel sponge assay; this inhibition was mimicked by the SSTR3 agonist L-796778 and reversed by the SSTR3 antagonist BN81658, demonstrating involvement of SSTR3. In vitro experiments showed that somatostatin directly affected different endothelial cell line proliferation through a block of growth-factor-stimulated MAPK and endothelial nitric oxide (NO) synthase (eNOS) activities. BN81658 reversed somatostatin inhibition of cell proliferation, NO production, and MAPK activity, indicating that SSTR3 activation is required for the effects of somatostatin in vitro. Finally in vivo angiogenesis assays demonstrated that eNOS inhibition was a prerequisite for the antiangiogenic effects of somatostatin, because high concentrations of sodium nitroprusside, an NO donor, abolished the somatostatin effects. In conclusion, we demonstrate that somatostatin is a powerful antitumor agent in vivo that inhibits tumor angiogenesis through SSTR3-mediated inhibition of both eNOS and MAPK activities.

A novel mechanism for the melatonin inhibition of testosterone secretion by rat Leydig cells: reduction of GnRH-induced increase in cytosolic Ca2+.

The site of inhibition, by melatonin, of GnRH-dependent testosterone secretion was investigated in adult rat Leydig cells cultured in vitro. The various effects downstream of the binding of GnRH to its own receptor were isolated and mimicked by specific drugs. Testosterone secretion was then evaluated after 3 h stimulation with GnRH, thapsigargin (1 microM), phorbol-12-myristate-13-acetate (100 nM), arachidonic acid (20 microM), and ionomycin (1 microM) in the presence or absence of melatonin (215 nM). The effect of melatonin on the GnRH-induced changes in cytoplasmic calcium concentration ([Ca(2+)](i)) was also studied, using Fura-2 as fluorescent Ca(2+) indicator. Melatonin attenuated the increase in [Ca(2+)](i) and inhibited the testosterone secretion induced by GnRH, but not that induced by ionomycin. Both ionomycin and thapsigargin potentiated GnRH-induced testosterone secretion; however, ionomycin, but not thapsigargin, partially prevented the inhibitory effect of melatonin on cells stimulated with GnRH. The effect of melatonin was probably dependent on the binding of melatonin to its Gi-protein-coupled receptor, as the inhibitory effect on GnRH-induced secretion was supressed in cells pretreated with pertussis toxin in a concentration of 180 ng/ml for 20 h. Assay of 17-hydroxy-progesterone showed that, irrespective of the treatment, cells cultured with melatonin secreted greater amounts than controls. We conclude that melatonin reduces GnRH-induced testosterone secretion by 1) decreasing [Ca(2+)](i), through impairment of the GnRH-dependent release of Ca(2+) from intracellular stores and 2) blocking 17-20 desmolase enzymatic activity, an effect that occurs irrespective of changes in [Ca(2+)](i).

Nitric oxide and GABAA receptor function in the rat cerebral cortex and cerebellar granule cells.

The aim of the present work was to investigate the mechanism by which the diffusible factor nitric oxide regulates GABAA receptor function in the brain. The effect of nitric oxide on GABAA receptor function has been studied in two different neuronal preparations: rat cerebral cortex microsacs and rat cerebellum granule cells in culture. In the first case, GABA-stimulated 36Cl-accumulation was studied as an index of GABAA receptor function. The maximal rate of GABA-stimulated 36Cl- accumulation (Vmax) was reduced by treatment of microsacs with nitric oxide chemical donors such as sodium nitroprusside (-26%) and S-nitroso-acetyl-penicillamine (-11%). The greater effect of the former agent is due to an additional interference by its breakdown products. The biochemical precursor L-arginine (1 mM) produced the same Vmax decrease as S-nitroso-acetyl-penicillamine. This effect was reversed by a nitric oxide synthase blocker and appears truly nitric oxide mediated. The action of nitric oxide in this system does not seem to imply cyclic GMP formation. GABAA receptor function was studied by whole-cell patch-clamp in rat cerebellum granule cells in culture. In this case, L-arginine (100 microM) profoundly reduced the Cl- current elicited by 10 microM GABA and its effect subsided following washing out. The effect of L-arginine was observed almost exclusively on the rapidly desensitizing component of the GABA-activated current. The action of L-arginine was blocked by a protein kinase G inhibitor and mimicked by its activators. Thus, it appears that this effect in these cells involves nitric oxide formation, cyclic GMP accumulation and protein kinase G-catalysed phosphorylation of GABAA receptor.

Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro.

OBJECTIVE: Somatostatin is a powerful inhibitor of hormone secretion and cell proliferation. Treatment with somatostatin analogs in humans causes a reduction in size and secretory activity of some endocrine tumors, including somatotropic pituitary adenomas. Less studied are the effects of somatostatin agonists on non-functioning pituitary adenomas (NFPAs). In this study we characterized the effects of somatostatin and its analog lanreotide on the proliferation of NFPAs in vitro and the intracellular mechanisms involved. DESIGN: Twenty-three NFPA post-surgical specimens were analyzed for somatostatin receptor (SSTR) expression and 12 of them were cultured in vitro to study somatostatin's effects on cell proliferation, assessed by means of [(3)H]thymidine uptake, and the intracellular signaling. RESULTS: One or more SSTR subtypes were expressed in 90% of the adenomas tested. Somatostatin and lanreotide treatment inhibited phorbol myristate acetate (PMA)-induced cell proliferation. Vanadate pretreatment reversed somatostatin and lanreotide inhibition of PMA-induced DNA synthesis suggesting an involvement of tyrosine phosphatase in this effect. In the only adenoma tested, somatostatin directly induced a tyrosine phosphatase activity. Somatostatin and lanreotide caused also a significant inhibition of voltage-sensitive calcium channel activity induced by 40mmol/l K(+) depolarization in microfluorimetric analysis. CONCLUSIONS: These data show that somatostatin and lanreotide inhibit human NFPA cell proliferation in vitro, and suggest that activation of tyrosine phosphatases and inhibition of the activity of voltage-dependent calcium channels may represent intracellular signals mediating this effect.

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein.

Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.

Prion protein fragment 106-126 induces a p38 MAP kinase-dependent apoptosis in SH-SY5Y neuroblastoma cells independently from the amyloid fibril formation.

Prion diseases are neurodegenerative disorders of the central nervous system of humans and animals, characterized by spongiform degeneration of the central nervous system, astrogliosis, and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (prion protein, PrP(C)) into an altered isoform (PrP(Sc)) has been proposed to represent the causative event responsible for these diseases. The peptide corresponding to the residues 106-126 of PrP sequence (PrP106-126) is largely used to explore the neurotoxic mechanisms underlying the prion diseases. We investigated the intracellular signaling responsible for PrP106-126-dependent cell death in the SH-SY5Y human neuroblastoma cell line. In these cells, PrP106-126 treatment induced apoptotic cell death and the activation of caspase-3. The p38 MAP-kinase blockers (SB203580 and PD169316) prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 activation. However, whether the neuronal toxicity of PrP106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. In this study, we correlated the structural state of this peptide with its neurotoxicity. We show that the two conserved glycines in position 114 and 119 prevent the peptide to assume a structured conformation, favoring its aggregation in amyloid fibrils. The substitution of both glycines with alanine residues (PrP106-126AA) generates a soluble nonamyloidogenic peptide, that retained its toxic properties when incubated with neuroblastoma cells. These data show that the amyloid aggregation is not necessary for the induction of the toxic effects of PrP106-126.

Somatostatin receptor 1 (SSTR1)-mediated inhibition of cell proliferation correlates with the activation of the MAP kinase cascade: role of the phosphotyrosine phosphatase SHP-2.

The mitogen activated protein (MAP) kinase cascade represents one of the major regulator of cell growth by hormones and growth factors. However, although the activation of this intracellular pathway has been often regarded as mediator of cell proliferation, in many cell types the increase in MAP kinase (also called extra-cellular signal regulated kinase: ERK) activity may result in cell growth arrest, depending on the length or the intensity of the stimulation. In this review we examine recent data concerning the effects of somatostatin on the MAP kinase cascade through one of its major receptor subtype, the somatostatin receptor 1 (SSTR1), stably expressed in CHO-K1 cells. Somatostatin inhibits the proliferative effects of basic FGF (bFGF) in CHO-SSTR1 cell line. However, in these cells, somatostatin robustly activates the MAP kinase and augments bFGF-induced stimulation of ERK. We show that the activation of ERK via SSTR1 is mediated by the betagamma subunit of a pertussis toxin-sensitive G-protein and requires both the small G protein Ras and the serine/threonine kinase Raf-1. Moreover the phosphatidyl inositol-3kinase and the cytosolic tyrosine kinase c-src participate in the signal transduction regulated by SSTRI to activate ERK, as well as it is involved the protein tyrosine phosphatase (PTP) SHP-2. Previous studies have suggested that somatostatin-stimulated PTP activity mediates the growth inhibitory actions of somatostatin, in CHO-SSTR1 cells. Thus, the activation of SHP-2 by SSTR1 may mediate the antiproliferative activity of somatostatin. SHP-2 may. in turn, regulate the activity of kinases upstream of ERK that require tyrosine dephosphorylation to be activated, such as c-src. Finally, the synergism between somatostatin and bFGF in the activation of ERK results in an increased expression of the cyclin-dependent kinase inhibitor p21cip/WAF1 as molecular effector of the antiproliferative activity of somatostatin.

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106-126.

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP(C)) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP(Sc)) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP(Sc) is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106-126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP(Sc), retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106-126 in different cellular models. We report that the PrP 106-126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH(3) cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106-126, while, in the cortical neurons and in the GH(3) cells, the same treatment causes a blockade of these channels causing a toxic effect.

Characterization of two central AMPA-preferring receptors having distinct location, function and pharmacology.

The existence of a pharmacological heterogeneity among the glutamate receptors sensitive to (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) has been investigated in the adult rat central nervous system (CNS). AMPA stimulated [3H]noradrenaline ([3H]NA) release from hippocampal synaptosomes (pD2 = 4.58) and the production of cGMP in cerebellar slices (pD2 = 7.75). The AMPA effects in the two systems were tested against several glutamate receptor antagonists including 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitro-quinoxaline-2,3-dione (DNQX), L-glutamate diethylester (GDEE), gamma-D-glutamyl-glycine (GDGG) and gamma-D-glutamyl-aminomethylsulphonate (GAMS). In both systems the AMPA effect was equally sensitive to CNQX or DNQX. However, while the AMPA-evoked increase of [3H]NA release from presynaptic terminals was not affected by GAMS, GDGG or GDEE, the postsynaptic cGMP response was prevented by GDGG and GDEE. It is concluded that rat hippocampus and cerebellum possess, respectively, presynaptic and postsynaptic AMPA-sensitive receptors involved in different functions and endowed with diverse pharmacological properties.

Serotonergic inhibition of the mossy fibre--granule cell glutamate transmission in rat cerebellar slices.

The glutamatergic mossy fibre-->granule cell pathway has been investigated in rat cerebellar slices. Exposure to 35 mM KCl, a concentration of K+ known to elicit Ca(2+)-dependent releases of excitatory amino acids from cerebellar slices, raised cGMP levels. The cGMP response was decreased in a concentration-dependent manner by D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) and by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) indicating the involvement of ionotropic glutamate receptors of both the N-methyl-D-aspartate (NMDA) and the non-NMDA type. The K(+)-evoked production of cGMP was potently inhibited (EC50 = 1.21 nM) by 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI), a selective 5-HT2 receptor agonist. The effect of DOI (0.01 microM) was antagonized by 0.03 microM of the 5-HT2 receptor antagonists ketanserin and methiothepin. At concentrations higher than 0.1 microM, both antagonists increased on their own the cGMP response elicited by high-K+. This effect was insensitive to tetrodotoxin. It had been previously shown that rat mossy fibre endings release glutamate upon depolarization and that such release can be inhibited by activation of 5-HT2 receptors sited on the mossy fibre endings. Altogether the available data suggest the following conclusions: a) the glutamate/aspartate endogenously released in cerebellar slices during K+ depolarization increase cGMP synthesis through the activation of both NMDA and non-NMDA receptors; b) a portion of the cGMP response can be prevented by 5-HT2 receptor activation and may reflect the activity of the mossy fibre--granule cell pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Polydeoxyribonucleotides enhance the proliferation of human skin fibroblasts: involvement of A2 purinergic receptor subtypes.

It is well-known that nucleotides, nucleosides and purine/pyrimidine bases enhance cell proliferation in vitro. Nevertheless, the molecular mechanisms involved in this mitogenic activity is still controversial, since these compounds are reported both to synergize with growth factor, and to act directly on purinergic receptor inducing per se a proliferative response. It was suggested that cell growth enhancement could be mediated by the A2 purinergic receptor activation. Here we report that a polydeoxyribonucleotide (PDRN) and adenosine are able to increase, the growth rate of human skin fibroblasts in primary cultures. The proliferative activity exerted by PDRN was significantly counteracted by the A2 antagonist 3, 7-Dimethyl-1-propargylxanthine (DMPX), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (PD 116,948, DPCPX). Accordingly, the trophic action of PDRN was mimicked by the A2 agonist N6-[2-(3,5-Dimethoxyphenyl)-2-(methylphenyl)-ethyl]adenosine (DPMA), while the A1 agonist N6-Cyclopenthyladenosine (CPA) did not show any effect. In microfluorimetric studies, we observed that PDRN and adenosine increased the concentration of cytosolic calcium ions. The PDRN-evoked calcium rise was dose-dependent and DMPX sensitive. Taken together, our results suggest that PDRN may operate as a pro-drug providing the cultured cells with an effective amount of mitogenic deoxyribonucleotides, deoxyribonucleosides and bases; moreover, cell proliferation enhancement that has been induced by PDRN seems to be mediated, at least in part, by the activation of purinergic receptors of the A2 subtype.

[Somatostatinergic control of Kaposi's sarcoma growth through the inhibition of angiogenesis]. / La somatostatina inibisce la crescita di sarcoma di Kaposi in topi nudi mediante l'inibizione della neoangiogenesi.

Somatostatin and its analogs are active in the inhibition of the proliteration of sst receptor positive endocrine neoplasms, however their activity and mechanism in non-endocrine tumors is not clear. Somatostatin effectively inhibited the growth of a Kaposi's sarcoma xenograft in nude mice, yet in vitro the tumor cells did not express any known somatostatin receptors and were not growth inhibited by somatostatin. Histological examination revealed limited vascularization in the somatostatin treated tumors as compared to the controls. Somatostatin was a potent inhibitor of angiogenesis in an in vivo assay. In vitro, somatostatin inhibited endothelial cell growth and invasion. Migration of monocytes, important mediators of the angiogenic cascade, was also inhibited by somatostatin. Both cells types expressed somatostatin receptor mRNAs. These data demonstrate that somatostatin is a potent anti-tumor angiogenesis compound directly affecting both endothelial and monocytic cells. The debated function of somatostatin in tumor treatment and the design of therapeutic protocols should be reexamined considering these data.

[The activation of the phosphotyrosine phosphatase eta is responsible for the somatostatin inhibition of PCCl3 thyroid cell proliferation]. / L'attivazione della fosfotirosino fosfatasi eta è responsabile dell'inibizione della proliferazione delle cellule tiroidee PCC13 ad opera della somatostatin.

BACKGROUND: This study was aimed to identify possible intracellular effectors of the somatostatin (SST) antiproliferative activity, in PCCl3 thyroid cells. METHODS: To prove the involvement of r-PTPeta in SST's effect, we studied th proliferative activity of subclones of PCCl3 cells that do or do not express this PTP. RESULTS: SST inhibited PCCl3 TSH+insulin-dependent cell proliferation through the induction of a phosphotyrosine phosphatase (PTP) activity, detected using the synthetic substrate pNPP (+150%, p<0.01). Conversely, PCCl3 cells stably expressing the v-mos oncogene (PCmos) were completely insensitive to SST antiproliferative effects due to the incapability of SST to increase PTP activity, that correlated with the abolishment of the expression of the receptor-like PTP, r-PTPeta. In the cells in which r-PTPeta was transfected (PCmos/ PTPeta) SST inhibited cell proliferation showing a dose-dependence similar to that observed in PCCl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTPeta did not restore the responsivity to SST. Also in PCmos/PTPeta cells SST, treatment increased membrane PTP activity. CONCLUSIONS: SST inhibition of PCC13 cell proliferation requires the activation of r-PTPeta.

Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction.

To define the mechanisms by which hPrP90-231 induces cell death, we analyzed its interaction with living cells and monitored its intracellular fate. Treatment of SH-SY5Y cells with fluorescein-5-isothiocyanate (FITC)-conjugated hPrP90-231 caused the accumulation of cytosolic aggregates of the prion protein fragment that increased in number and size in a time-dependent manner. The formation of large intracellular hPrP90-231 aggregates correlated with the activation of apoptosis. hPrP90-231 aggregates occurred within lysotracker-positive vesicles and induced the formation of activated cathepsin D (CD), indicating that hPrP90-231 is partitioned into the endosomal-lysosomal system structures, activating the proteolytic machinery. Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis. Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis. By confocal microscopy analysis of lucifer yellow (LY) intracellular partition, we show that hPrP90-231 accumulation induces lysosome destabilization and loss of lysosomal membrane impermeability. In fact, although control cells evidenced a vesicular pattern of LY fluorescence (index of healthy lysosomes), hPrP90-231-treated cells showed diffuse cytosolic fluorescence, indicating LY diffusion through damaged lysosomes. In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.

Intracellular transducing mechanisms coupled to brain somatostatin receptors.

This review systematically analyses recent knowledge of the biology of somatostatin receptors. Indeed, since the molecular cloning of five somatostatin receptors in 1992, a growing bulk of scientific data has been produced regarding the cell type localization, the physiological role and the biochemical intracellular pathways activated by the single somatostatin receptors. The aim of this review is to present all these data, also discussing the conflicting evidence that has been reported, to further simulate research efforts in the field.

Effect of nitric oxide donors on GABA uptake by rat brain synaptosomes.

The effect of nitric oxide donors and L-arginine on the uptake of GABA was studied in synaptosomes purified from rat brain. The neurotransmitter uptake was significantly reduced by S-nitrosoacetylpenicillamine and by sodium nitroprusside, although in this case to a lesser extent. A slight inhibitory effect was found preincubating rat brain synaptosomes with 1 mM L-arginine as well. The S-nitrosoacetylpenicillamine effect gradually disappeared with decomposition of the substance by exposure to light. The nitric oxide effect appears to be mainly due to a decrease in the V for synaptosomal GABA uptake and seems to be related to a partial collapse of nerve endings ionic gradients. Functionally, it could result over time in a reduced availability of GABA at the synapses involved.