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A mole of Mercury was suitably treated by ultrasound in order to generate in it the same conditions of local Lorentz invariance violation that were generated in a sonicated cylindrical bar of AISI 304 steel and that are the cause of neutron emission during the sonication. After 3 min, part of the mercury turned into a solid material which turned out to contain isotopes having a different mass (higher and lower) with respect to the isotopes already present in the initial material (mercury). These transformations in the atomic weight without gamma production above the background are brought about during Deformed Space-Time reactions. We present the results of the analyses performed on samples taken from the transformation product. The analyses have been done in two groups, the first one using five different analytical techniques: ICP-OES, XRF, ESEM-EDS, ICP-MS, INAA. In the second group of analyses, we used only two techniques: INAA and ICP-MS. The second group of analyses confirmed the occurring of the transformations in mercury. Read More: http://www.worldscientific.com/doi/abs/10.1142/S0217979217501685

At interfaces between complex oxides it is possible to generate electronic systems with unusual electronic properties, which are not present in the isolated oxides. One important example is the appearance of superconductivity at the interface between insulating oxides, although, until now, with very low Tc. We report the occurrence of high Tc superconductivity in the bilayer CaCuO2/SrTiO3, where both the constituent oxides are insulating. In order to obtain a superconducting state, the CaCuO2/SrTiO3 interface must be realized between the Ca plane of CaCuO2 and the TiO2 plane of SrTiO3. Only in this case can oxygen ions be incorporated in the interface Ca plane, acting as apical oxygen for Cu and providing holes to the CuO2 planes. In addition, a detailed hole doping spatial profile can be obtained by scanning transmission electron microscopy and electron-energy-loss spectroscopy at the O K edge, clearly showing that the (super)conductivity is confined to about 1–2 CaCuO2 unit cells close to the interface with SrTiO3. The results obtained for the CaCuO2/SrTiO3 interface can be extended to multilayered high Tc cuprates, contributing to explaining the dependence of Tc on the number of CuO2 planes in these systems.

At interfaces between complex oxides it is possible to generate electronic systems with unusual electronic properties, which are not present in the isolated oxides. One important example is the appearance of superconductivity at the interface between insulating oxides, although, until now, with very low T(c). We report the occurrence of high T(c) superconductivity in the bilayer CaCuO(2)/SrTiO(3), where both the constituent oxides are insulating. In order to obtain a superconducting state, the CaCuO(2)/SrTiO(3) interface must be realized between the Ca plane of CaCuO(2) and the TiO(2) plane of SrTiO(3). Only in this case can oxygen ions be incorporated in the interface Ca plane, acting as apical oxygen for Cu and providing holes to the CuO(2) planes. A detailed hole doping spatial profile can be obtained by scanning transmission electron microscopy and electron-energy-loss spectroscopy at the O K edge, clearly showing that the (super)conductivity is confined to about 1-2 CaCuO(2) unit cells close to the interface with SrTiO(3). The results obtained for the CaCuO(2)/SrTiO(3) interface can be extended to multilayered high T(c) cuprates, contributing to explaining the dependence of T(c) on the number of CuO(2) planes in these systems.

Background/Aims : Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC). Methods : HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70 S6K activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and α1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively. Results : Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 μM, with a maximal effect at 1000 μM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70 S6K activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na + /H + exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 μM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 μM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor β1 by 54% and 92%, respectively, as well as TGF β1 -induced α1(I) procollagen mRNA expression. Results : Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

The presence of large mucilaginous masses, referred to as mucilage or with the traditional term “dirty sea”, has led to serious problems for the tourism and fishing industries along the coast of the northern Adriatic Sea. Using a cable-guided telecamera, these masses were observed in situ from the water surface along the water column to the sea bottom, with the aim of better understanding the appearance of the mucilage, its formation and dispersion and the processes originated by phytoplankton involved in this phenomenon. Furthermore, we sought information to help explain the fact that the appearance of mucilage does not tend to lead to anoxia in the surrounding water and that the mucilage seemed to containhuge amounts of iron. The chemical-physical characteristics of mucilage samples and samples of the surrounding water were studied. Further studies of their composition were carried out to determine the levels of organic matter, organic carbon, carbohydrates and heavy metals.

BACKGROUND: Insulin resistance is a significant risk factor for hepatic fibrosis in patients with both non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C (CHC), either directly or by favouring hepatic steatosis. Several methods are available to assess insulin resistance, but their impact on this issue has never been evaluated. AIMS: To determine the relative contribution of steatosis, metabolic abnormalities and insulin resistance, measured by different basal and post-load parameters, to hepatic fibrosis in CHC and in NAFLD patients. METHODS: In 90 patients with CHC and 90 pair-matched patients with NAFLD, the degree of basal insulin resistance (by the homeostasis model assessment, (HOMA)) and post-load insulin sensitivity (by the oral glucose insulin sensitivity (OGIS) index) was assessed, together with the features of the metabolic syndrome according to Adult Treatment Panel III definition. Data were correlated with hepatic histopathology. RESULTS: The prevalence of basal insulin resistance (HOMA values >75th percentile of normal) was 23.3% in CHC patients and 57.8% in NAFLD, but it increased to 28.8 and 67.8% when measured by post-load insulin resistance (OGIS

Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells. IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC). This study evaluates the potential roles of these hormones in the development of liver fibrosis. Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue. Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively. Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis. Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation. Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin. Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway. IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism. In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.

Background/Aims: Oxidative stress is associated with liver fibrosis in vivo and with hepatic stellate cell (HSC) activation in vitro , but the intracellular mechanisms mediating these effects are mostly unknown. The Na + /H + exchanger plays a key role in regulating the cell cycle, and is involved in HSC proliferation. Its role in different HSC features, such as collagen accumulation, is still unknown. We thus evaluated if the Na + /H + exchanger modulates the fibrogenic effect of oxidative stress in rat HSC. Methods: HSC were incubated with 0.1 mM ferric nitrilotriacetate complex (FeNTA). Intracellular hydroperoxides and malonildialdehyde (MDA) levels in the culture media were measured by the dichlorofluorescein and TBARS method, respectively. Intracellular pH and Na + /H + exchanger activity were measured using the fluorescent dye BCECF. Cell proliferation was measured by immunohistochemistry for bromodeoxyuridine incorporation. Collagen type I accumulation in the culture media was measured by ELISA. Results: HSC incubation with FeNTA resulted in a significant production of intracellular hydroperoxides and MDA, associated with increased Na + /H + exchange activity and baseline intracellular pH (pHi). Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium. All these effects were reversed by the antioxidant resveratrol and by the Na + /H + exchanger inhibitor amiloride. Conclusions: This study indicates that the Na + /H + exchanger might represent a common mediator of the different effects induced by oxidative stress on HSC. The reduction in cell proliferation and collagen synthesis induced by amiloride could represent a new therapeutic challenge in liver fibrosis.

Background & Aims: The Na + /H + exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na + /H + exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na + /H + exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na + /H + exchanger by PDGF in HSCs. Methods: The activity of the Na + /H + exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na + /H + exchange protein expression was evaluated by means of Western blot. Results: PDGF induced a significant increase in the activity of the Na + /H + exchanger without modifying protein expression. Inhibition of the calcium/calmodulin– and protein kinase C–dependent pathways resulted in a significant inhibition of both Na + /H + exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na + /H + exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na + /H + exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase– and of phosphatidylinositol 3-kinase–dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na + /H + exchanger. Conclusions: Activation of the Na + /H + exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C–dependent pathways. PDGF-induced HSC proliferation is mediated by Na + /H + exchange–dependent and –independent pathways. GASTROENTEROLOGY 1999;116:1155-1166

Oxidative stress is associated with liver fibrosis and with hepatic stellate cell (HSC) activation in vivo. However, it remains controversial whether oxidative stress contributes to HSC activation either directly or through a paracrine stimulation by damaged hepatocytes. A medium containing products released from cells undergoing oxidative stress was obtained after incubation of hepatocytes with (HCM/Fe) or without (HCM) 0.1 mmol/L ferric nitrilotriacetate complex (FeNTA). Exposure of HSC to HCM/Fe for 24 hours significantly increased the number of proliferating HSC compared with HCM and to controls at all dilutions tested. The simultaneous coincubation of HSC with HCM/Fe and desferrioxamine (50 micromol/L) did not reduce the observed increase in cell proliferation, thus excluding a role for eventually contaminating iron in HCM/Fe. HCM/Fe induced also a significant increase in collagen type I accumulation in HSC culture media. To study the cellular mechanism underlying HCM/Fe effects, we evaluated the activity of the Na+/H+ exchanger, which plays a role in regulating HSC proliferation. The incubation of HSC for 24 hours with HCM/Fe significantly increased baseline intracellular pH (pHi) and Na+/H+ exchanger activity, indicating a plausible role of this antiport in mediating cell response. In conclusion, hepatocytes undergoing oxidative stress release factors which are fibrogenic for HSC, thereby, confirming what has been only hypothesized in vivo. In addition, HSC proliferation is associated with changes in the Na+/H+ exchanger activity, thus providing a useful target for the evaluation of inhibitors of this pathway for the treatment of hepatic fibrosis.

By using oligonucleotide hybridization, restriction endonuclease analysis and direct sequencing of amplified genomic DNA, we have been able to characterize 18 different mutations in the beta-globin genes of 161 beta-thalassemia homozygotes and 107 beta-thalassemia heterozygotes from Turkey (429 beta-thalassemia chromosomes). Previous studies dealing with beta-thalassemia in Mediterranean countries have shown that, in most Mediterranean populations, only a few mutations are prevalent. In contrast, beta-thalassemia in Turkey does not seem to be associated with a few predominant mutations. The six most frequent alleles, IVS-I-110 (G----A), IVS-I-6(T----C), FSC-8 (-AA), IVS-I-1(G----A), -30(T----A) and FSC-5 (-CT), account for only 69.3% of the disease genes; indeed, all 26 mutations assayed represent 85.8% of the disease genes, confirming the considerable molecular heterogeneity of beta-thalassemia among Turks, and indicating the possible presence of rare, previously undefined, mutations in the population. Two mutations observed in this study, IVS-I-116 (T----G) and Cd44(-C), have not been reported in the Turkish population to date. Since preventive medical services, such as genetic counseling and prenatal diagnosis, are greatly improved by detailed knowledge of the molecular pathology of beta-thalassemia, we strongly believe that the presented data will facilitate the intended establishment of a prenatal diagnosis center, based on DNA analysis, in Turkey.

Non-invasive techniques are being developed to assess the severity of liver disease. Haemodynamic changes in the hepatic circulation during the development of liver disease can be evaluated with contrast-enhanced ultrasound.To evaluate the possible correlation between ultrasound contrast-agent transit times and different stages of chronic hepatitis C.Sixteen patients with clinically evident hepatitis C virus-related cirrhosis, 22 non-cirrhotic patients with chronic hepatitis C and 14 controls with no clinical evidence of liver disease were studied.Contrast-enhanced hepatic ultrasonography was performed with a sulphur hexafluoride-filled microbubble contrast agent, and time curves of hepatic vein signal intensity were analysed to determine the time of enhancement onset (hepatic vein arrival time) and peak enhancement (hepatic vein peak enhancement).Hepatic vein arrival time in cirrhotic patients was significantly shorter (p0.001) than in non-cirrhotic patients and controls. Within the group with chronic hepatitis C, METAVIR scores of fibrosis and necro-inflammatory changes had no significant effect on hepatic vein arrival times.Analysis of the time of onset of ultrasound contrast enhancement of the hepatic vein appears to be a simple, non-invasive method for reliably excluding cirrhosis with signs of portal hypertension, but not for assessing the severity of either chronic hepatitis C or cirrhosis.

Experimental evidence indicates that reactive oxygen species (ROS) are involved in the development of hepatic fibrosis; they induce hepatic stellate cells (HSC) proliferation and collagen synthesis. To address the role of matrix metalloproteinase (MMP)-2 in promoting HSC proliferation during hepatic injury, we investigated whether oxidative stress modulates the growth and invasiveness of HSC by influencing MMP-2 activation. Cell invasiveness and proliferation, which were studied using Boyden chambers and by counting cells under a microscope, were evaluated after treatment with a superoxide-producing system, xanthine plus xanthine oxidase (X/XO), in the presence or absence of antioxidants and MMP inhibitors. Expression and activation of MMP-2 were evaluated via gel zymography, immunoassay, and ribonuclease protection assay. The addition of X/XO induced proliferation and invasiveness of human HSC in a dose-dependent manner. The addition of antioxidants as well as MMP-2-specific inhibitors impaired these phenomena. X/XO treatment increased MMP-2 expression and secretion appreciably and significantly induced members of its activation complex, specifically membrane-type 1 MMP and tissue inhibitor metalloproteinase 2. To study the intracellular signaling pathways involved in X/XO-induced MMP-2 expression, we evaluated the effects of different kinase inhibitors. The inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3-kinase (PI3K) abrogated X/XO-elicited MMP-2 upregulation and completely prevented X/XO-induced growth and invasiveness of HSC. In conclusion, our findings suggest that MMP-2 is required for the mitogenic and proinvasive effects of ROS on HSC and demonstrate that ERK1/2 and PI3K are the main signals involved in ROS-mediated MMP-2 expression.

Background B Aims: Hepatic stellate cell (HSC) proliferation is a key event in the development of liver fibrosis. In many liver diseases, HSCs are exposed to inflammatory cytokines, reactive oxygen species, and bile acids. Although inflammatory cytokines and reactive oxygen species are known to promote proliferation of HSCs, nothing is known about the effects of bile acids on HSC proliferation or apoptosis. The aim of this study was to investigate the effects of bile acids on HSC proliferation. Methods: HSCs were exposed to bile acids with different hydrophobicity (5-200 mu mol/L). HSC proliferation and cell cycle-related events were assessed by bromodeoxyuridine incorporation, cell counting and proliferating cell nuclear antigen and cyclin E expression, apoptosis by caspase-3 activity assay, immunocytochemistry for active caspase-3 and acridine orange staining, and activation of signal transduction pathways by Western blot using phospho-specific antibodies. Uptake of bile acids was investigated using fluorescent bile acids. Results: All bile acids, at concentrations > 25 mu mol/L, induce a 2.5- to 3-fold increase in HSC proliferation via activation of the epidermal growth factor receptor. Bile acid-induced proliferation is mediated by activation of a protein kinase C/extracellular signal-regulated kinase/p70(S6K_) dependent pathway. Bile acids did not induce apoptosis in HSCs. HSCs do not take up fluorescent bile acids and do not express the bile acid importer ntcp. Conclusions: Bile acids at levels reached in cholestatic conditions are an independent profibrogenic factor. Bile acids induce HSC proliferation via the activation of the epidermal growth factor receptor, whereas HSCs are protected against bile acid-induced apoptosis by excluding bile acids.

Background/Aims : The regulation of three major intracellular signalling protein kinases was investigated in two models of liver injury leading to hepatic fibrosis, dimethylnitrosamine administration (DMN) and bile duct ligation (BDL). Methods : Extracellular signal-regulated kinases (ERK)1/2, c-Jun terminal kinase (JNK) and p70S6-kinase (p70 S6K ) were studied in vivo in the whole liver, in liver sections and in isolated hepatocytes, cholangiocytes and hepatic stellate cells (HSC). Results : In the whole liver, activation of these kinases occurred with a different kinetic pattern in both models of liver injury. By immunohistochemistry and Western blot in isolated cells, phosphorylated kinases were detected in proliferating cells (i.e. hepatocytes and cholangiocytes after DMN and BDL, respectively), in addition to stellate-like elements. ERK1/2, JNK and p70 S6K activation was associated with hepatocytes proliferation after DMN, while JNK activation was not associated with cholangiocytes proliferation after BDL. In HSC isolated from injured livers, protein kinases were differentially activated after BDL and DMN. Kinases activation in HSC in vivo preceded cell proliferation and alpha-smooth muscle actin appearance, a marker of HSC transformation in myofibroblast-like cells, and collagen deposition. Conclusions : Our findings indicate that these kinases are coordinately regulated during liver regeneration and suggest that their modulation could be considered as a future therapeutic approach in the management of liver damage.

Background & Aims: The ligand-dependent transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is expressed in hepatic stellate cells (HSC), and its transcriptional activity is reduced during cell transdifferentiation in culture. PPARγ transcriptional activation decreases platelet-derived growth factor–induced proliferation and inhibits α-smooth muscle actin expression in cultured HSC. The aim of our study was to evaluate whether oral administration of synthetic PPARγ ligands, thiazolidinediones (TZD), might affect collagen deposition in animal models of liver fibrosis. Methods: The effect of 2 TZD (pioglitazone or rosiglitazone) was tested on liver fibrosis induced in rats by either toxin administration (dimethylnitrosamine or carbon tetrachloride) or bile duct ligation. In vivo PPARγ activation was evaluated by gel shift assay using nuclear extracts from HSC isolated from control and treated rats. Results: Oral administration of TZD reduced extracellular matrix deposition and HSC activation in both toxic and cholestatic models of liver fibrosis. PPARγ-specific DNA binding was significantly impaired in nuclear extracts of HSC isolated from fibrotic rats compared with HSC from control rats. TZD administration restored PPARγ DNA binding in HSC nuclei. In vitro, TZD-induced PPARγ activation inhibited collagen and fibronectin synthesis induced by transforming growth factor (TGF)-β1 in human HSC, as measured by enzyme-linked immunosorbent assay and Northen blotting. TZD also reduced the TGF-β1–induced activity of a 3.5-kilobase procollagen type I promoter transfected in human HSC. Conclusions: These findings indicate that PPARγ activation in HSC retards fibrosis in vivo and suggest the use of TZD for the treatment of liver fibrosis. GASTROENTEROLOGY 2002;122:1924-1940

Background/Aims : Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC. Methods/Results : Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C. Conclusions : PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury.

Ethanol induces liver fibrosis by several means that include, among others, the direct fibrogenic action of acetaldehyde on hepatic stellate cells (HSC). However the mechanisms responsible for this effect are not well understood. In this communication we investigated signal transduction pathways triggered by acetaldehyde leading to upregulation of alpha2(I) collagen and fibronectin gene expression in human HSC. Run-on assays showed that acetaldehyde-enhanced transcription of these 2 genes as early as 2 hours, via de novo protein synthesis-independent and -dependent mechanisms. It also stimulated a time-dependent induction in phosphorylation of pp70(S6K) and extracellular-regulated kinase (1/2) (ERK1/2). These effects were completely prevented by calphostin C, a protein kinase C inhibitor. As expected, acetaldehyde-elicited ERK1/2 phosphorylation was inhibited by PD98059, a MEK inhibitor, but not by wortmannin, a PI3K inhibitor. On the other hand, both of these inhibitors partially inhibited phosphorylation of pp70(S6K) induced by acetaldehyde suggesting that its activation is ERK1/2- and PI3K-dependent. Acetaldehyde-elicited fibronectin and alpha2(I) collagen upregulation was inhibited by calphostin C. However, while PD98059, wortmannin and rapamycin (a pp70(S6K) inhibitor) completely abrogated alpha2(I) collagen upregulation, they had no effect on fibronectin expression. Overall, these data suggest that protein kinase C is an upstream component from which acetaldehyde signals are transduced to other pathways such as PI3K and ERK1/2. In addition, differential activation of these pathways is needed for the increase in fibronectin and alpha2(I) collagen gene expression induced by acetaldehyde in human HSC.

Background/Aims : The Na + /H + exchanger is involved in rat hepatic stellate cell (HSC) proliferation induced by platelet-derived growth factor (PDGF). We therefore evaluated in human HSC: (1) the mechanisms of intracellular pH regulation; (2) the relationship between Na + /H + exchange activation and cell proliferation induced by PDGF, insulin-like growth factor 1 (IGF-1) and insulin. Methods/Results : pH i regulation was mainly dependent on the activity of the Na + /H + exchanger, which was evaluated by measuring pH i recovery from an acute acid load. PDGF (25 ng/ml) gradually increased the activity of the Na + /H + exchanger which peaked at 18 h and remained stable until the 24th h. IGF-1 (10 nmol/l), but not insulin (100 nmol/l), slightly but significantly increased the activity of the Na + /H + exchanger. Amiloride (100 μ mol/l) and 20 μ mol/l 5- N -ethyl- N -isopropyl-amiloride completely inhibited HSC proliferation (evaluated by measurement of bromodeoxyuridine incorporation) induced by PDGF and IGF-1, but did not affect proliferation of HSC induced by insulin. Finally, IGF-1 did not modify the activity of the Na + /Ca 2+ exchanger. Conclusions : The Na + /H + exchanger is involved in HSC proliferation induced by PDGF and IGF-1, whereas the proliferative effect of insulin is mediated by intracellular pathways which are Na + /H + exchange-independent.

Background & Aims: The Na + /H + exchanger is the main intracellular pH (pH i ) regulator in hepatic stellate cells (HSCs) and plays a key role in regulating proliferation and gene expression. We evaluated the effect of specific inhibition of this exchanger on HSC proliferation and collagen synthesis in vivo and in vitro. Methods: Rat HSCs were incubated in the presence of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1, iron ascorbate (FeAsc), and ferric nitrilotriacetate solution (FeNTA) with or without the Na + /H + exchanger inhibitor 5- N -ethyl- N -isopropyl-amiloride (EIPA). pH i and Na + /H + exchanger activity, cell proliferation, and type I collagen accumulation were measured by using the fluorescent dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, by immunohistochemistry for bromodeoxyuridine, and by enzyme-linked immunosorbent assay, respectively. In vivo liver fibrosis was induced by dimethylnitrosamine administration and bile duct ligation (BDL) in rats treated or not treated with amiloride. Results: PDGF, FeAsc, and FeNTA increased Na + /H + exchange activity and induced HSC proliferation. TGF-β1 had no effect on the Na + /H + exchanger and was able, as for FeAsc and FeNTA, to induce type I collagen accumulation. EIPA inhibited all the effects determined by PDGF, FeAsc, and FeNTA and had no effect on TGF-β1–induced collagen accumulation. In vivo, amiloride reduced HSC proliferation, activation, collagen deposition, and collagen synthesis. Conclusions: The Na + /H + exchanger can play a key role in the development of liver fibrosis and in HSC activation in vivo. GASTROENTEROLOGY 2001;120:545-556

Using restriction endonuclease analysis, oligonucleotide hybridization, and direct sequencing of amplified genomic DNA, we characterized 11 different mutations in the DNA of 26 patients from Turkey homozygous for beta-thalassemia. We found that mutations IVS-1 nt110, IVS-1 nt6, and the frameshift at codon 8 were the most frequent. By direct sequencing we characterized two very rare mutations not previously reported in the Turkish population: a frameshift +1 at codons 9/10 and a nonsense mutation at codon 15.

Results: We demonstrate 1) That both core tumour and non-tumourderived sequences inhibit the global antiviral effects of IFNa on VSV and EMCV replication in Ratl and HuH7 cells and sensitize Ref cells to transformation, in cooperation with ras oncogene. 2) That tumour and nontumour derived cores show, however, a quite distinct pattern of biological effects on IFNa signaling: inhibition of global antiviral effect of IFNa in tumour-derived expressing cells is much less pronounced, compared with non tumour-derived and reference lb cores.Expression of tumour-derived, and not non-tumour-derived nor reference lb cores, induces transactivafion of 0interferon sensitive response element0 (ISRE); tumour-derived core also increases phosphorylation of Statl and Stat 2, compared with nontumour-derived and reference lb proteins.Tumour-derived core show a markedly increased transforming activity in REF cells, in cooperation with ras oncogene, compared with non-tumour-derived and reference lb cores. Conclusion: Our data 1) support a role for HCV core in the resistance to global antiviral effect of IFNa (despite ISRE activation) and cell transformation in REF cells. 2) demonstrate a distinct pattern of biological effects of tumour and non-tumour-derived cores on interferon signaling and cell transformation, correlated with the hypothesis of selection of core mutants in clonaly expanding liver cells. Data obtained with 3 additional tumour and non-tumour-derived core sequences will be discussed.