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2 P Genomic polymorphism of augmenter of liver regeneration in healthy individuals
BIOTECHNOLOGY
DIGEST LIUER MS 2002:34lSUPPL.1kA137
.P
3P
EFFECT OF AUGMENTER OF LIVER REGENERATION REGULATORYGENES
A NEW SUPPORT FOR HEPATOCYTE CULTURE BASED ON HOMOLOGOUS ACELLULAR MATRIX (HAhQ
ON APOPTOSIS
L. Polimeno, M Margiotta, G Manco, B Pesetti, R Francav~lla, S Morisco, E lerardi A Francavilla Dipartimento Pediatria Background Partial bepatectomy (PH) is followed by liver cell proliferation in order to reconstitute the physiologic hepatic mass This process is followed and balanced by an apoptotic stimulus aimed to control the regeneratwe spur Indeed, PH is followed by the activation of the genes controlling growth factors and anti apoptotic genes (bcl-2, bcl-xL) that is followed by a late activation of pro apoptosis genes (bad, bak, bax) ALR is a liver growth factor and its serum levels atier 70% PH in rats peaks after 18 hours and return to normal after the 24th hour Moreover, ALR induces the expression of mitochondrial genes followed by an up-regulation of the oxidative phosphorylation ALR is mostly localised in the mitochondrial inter-membrane space and similarly to other factors in that site (Cytocrome C) can be considered a marker of mttochondrial integrity. One of the biological processes character&d by the alteration ofthe mitochondrial permeability is apoptosis Aim to define the effect of ALR on the expression of the apopiosis regulatory genes in the rat model of 70% hepatectomy. Materials and methods Ten male Fisher rats (F344) of 180-200 grams of body weight underwent 70% partial hepatectomy Five animals were treated i p with ALRp (0.5 w/day in saline) and five with albumin at 24. 36. 48 and 60 hours post surxery. The rats were &r&d at 48 and 72 hours post surgely We have analysed the iiver tissue for the activation of the anti-aooototic fbcl-2) and the ore-aooutotic ibax and ICE) genesbv RTPCR Results Our data show t&t at tie 72nd l& t&e is a dteady red&& ofth;proapoptotic genes mRNA (ICE -7O%, bax -50%) and an increased expression ofthe antiapoptotic gene mRNA (bcl-2 +30%) Conclusions. ALR modifies the expression of genes that regulate apoptosis induced by 70% PH in rats We argue that proliferatmn does not depend just on the growth factors but also on factors that inhibit cell death We believe that the release m the cytoplasm ofthe ALR is the biological process by which the cell contrasts apoptosis by preserving the maochondrial integrity in a phase of increased energy needs such during cell probferation
2P
F P RUSSO “, S TOMATO, M CONCONI §,P PARNlGOTTO 5, R NACCARATO BURRA”
‘, P
‘DIPARTIMENTO DI SClENZE CHIRURGICHE E GASTROENTEROLOGICHE; 8DlPARTIMENTO DI SCIENZE FARMACEUTICHE Introduction. Maintenance of different hepatocyte function in monolayer culture is generally short-lived Exogenous extracellular matrix (ECM) can mantain hepatocyte tin&n for a longer time. Whilst purified ECM components may be useful experimentally, there may be clinically bio-compatibility problems, such as clotting induced by collagen A biological support iniluencing cell development, migration, proliferation, shape and metabolic function would be very useful. The aim of the study was to obtain an homologous acellular matrix in order to support rat hepatocytes in primary culture. Materials and methods The HAM was prepared the day before each experiment by multiples rat liver slices. Slices were taken in sterile water, deoxycholic acid sodium salt solution and then digested with DNase. Experiments were performed on 25 male Sprague-Dawley rats Each animals was anesthetized The abdomen was opened and the liver was perfused in situ through vena porta with collagenase Isolated cells were counted in 1’ 10 diluted trypan blue and seededon 40 polystrene wells, 20 collagen coated wells and 60 HAM coated wells. Tbe number of viable cells was assayed by the thiawlyl blue (h4TT) assay based on mitochondrial reduction reaction Results About 8x106 of intact rat hepatocytes were obtained by each rat liver by using collagenasedigestion. Cell viability was about 80% The optimal seeding concentration was 2-3x105 cells/cm2 This concentration was used on polystyrene wells (chosen as control) and on collagen and HAM coated wells The MTT assays were performed at 24 hours and then daily after seeding Thanks to a calibration curve it was possible to express the absorbance value of MTT assay as number of viable cells (VC). 89 333, 50 000, 18.666 VC on polysyrene wells, 182.000, 101 333, 62 666 VC on collagen coated wells, 195 333, II2 666, 71 333 VC on HAM coated wells at 24, 96, 144 hours respectively The positivity ofthe MTT demonstrated a high activity of mitochondrium and the histology (emathoxylin-rosin and toluidin blu) showed a normal morphology ofhepatocytes cultured in the HAM Conclusion- These preliminary results suggest that hepatocytes in this new model of acellular biological matrix find their nomu. situation in terms of support and have at least an equal Sun&al as collagen
IP GENOM,C POLYMORPHISM nEALTHY lNDlvlDUALS
OF AUGMENTER OF LIVER REGENERATION
IN
M Margiona*, L Polimeno*, G Manco*. B Pesett~‘. E Ierardi#, R Francavdla’, S Morisco*, A Francavilla’
EXPRESSION OF HEAT SHOCK PROTEINS IN RESPONSE TO THE OXIDATIVE STRESS IN PATIENTS WITH CHRONIC LIVER DISEASES F Terrac&no*, A. Fed&x*, T De Sirnone’, C. Tuccillo*, E Fmamore”, C Loguerc~a*,C Del Vecchio Blanco, F Galdiero” ‘Gastroentemlogy School - 2nd University of Naples; ‘Institute ofMicrobiology - 2nd Unwersity of Naples
Introduction and Aim The genomic polymorphism, that is based on mutations ofDNA sequences, plays a key role both in the definition ofindividual phenotypic diversity and m disease ‘s predisposition The Augmenter of Liver Regeneration (ALR) gene. a well known hepatic growth factor, is located on the short arm ofchromosome 16 in humans and is composed of a sesuence of 2496 base pair It has been demonstrated that ALR play a crucial role in the mitochbndria bmgenesis DkA mutation of this factor may lead to functional mutation ofthe protein that may be of great imponance in diseases for which mitochondria metabolism is crucial (myastenic, astenospermic, neurological syndromes and hemocromatosis). At present little is known on the factors that control ALR gene expression and on the molecular pattern characteristic of the genomic polymorphism in healthy individuals therefore we have planned to explore this topic in our geographical area Material and methods. The genonuc DNA of 30 healthy indwlduals has been extracted from peripheric blood, amplified by nested PCR using specific pruners corresponding to two sequences of the second and third exon of the ALR gene The PCR products, less than 200 bp, have been separated by electrophoresis using the Smgle Strand Conformation Polvmomhism (SSCP) and silver-stained Usine this method, the aenomic sequences develor a c&nplex stm&e a&ding with their base &position with acharacter& electrophoretic pattern This method allows to recognise mutation extended to just a single base Results Our results, referred to the DNA sequences studied, have shown the electrophoretic pattern below reported In the healthy individuals of our geographic area the ALR genehave different allelic variations and the gene product keeps all the biological functions Conclusions. Our data, although preliminary on healthy individuals, represent for human the normal control to which refer for the ALR gene analysis in case of pathologies associated with mitochondrial metabolism dysfunction as yet not identitied
The family of Heat Shock Proteins HSP includes different forms on the basis of molecular weight These regulatory proteins are producted by cells of all organisms as response to stress. In human cells,HSP27 contribute to the stabilization of intracellular actin filaments and could play a regulatory role for the organization of the cytoskeleran Nitric oxlde~O),and particularly NO-thiol adducts(SH-NO),intluence the type and the entity of HSP expression Both HSP and NO affect genetranscription and apoptosis In experimental animals, HSP (60, 70, 90) were studied in relatmnship to cholestaas, as well as to NO and glutathione(GSH) production and limction. Several data documentedan increase of oxidative stress and a decreaseof GSH in patients with chronic liver damage We evaluated lymphocyte expression of HSP in relationship to SH-NO, glutathione-stransferase(GST) and markers of oxidative stress (malan,ldialdehyde.MA, and 4hydroxinonenal,4-HNE) plasma levels in patients with chronic liver disease PATIENTS- 16 with HCV-related biopsy-proven chronic hepatitis(CH), 16 with HCVrelated and 5 with alcoholic cirrhosis (10 Child A, I IChild B+C) Ten healthy subjects as control group METHODS HSP expression was evaluatedby Western blot, SH-NO by spectraphotometric method, GST, MDA and 4.HNE by commercial kits RESULTS HSP60 was positive m 5% of all patients globally cansldered and m none control HSPZ7,ahsentin controls, was expressed in 46,7% CH and 71.4% cmhotics (50% m Chdd A; 91% in Child B+C) SH-NO levels were increased in 88% of patients HSP27posrtive and in 30% of negative (mean *SD= 120 *23 vs 72 +25, p
Al37
DIGEST LIUER MS 2002:34lSUPPL.1kA137
.P
3P
EFFECT OF AUGMENTER OF LIVER REGENERATION REGULATORYGENES
A NEW SUPPORT FOR HEPATOCYTE CULTURE BASED ON HOMOLOGOUS ACELLULAR MATRIX (HAhQ
ON APOPTOSIS
L. Polimeno, M Margiotta, G Manco, B Pesetti, R Francav~lla, S Morisco, E lerardi A Francavilla Dipartimento Pediatria Background Partial bepatectomy (PH) is followed by liver cell proliferation in order to reconstitute the physiologic hepatic mass This process is followed and balanced by an apoptotic stimulus aimed to control the regeneratwe spur Indeed, PH is followed by the activation of the genes controlling growth factors and anti apoptotic genes (bcl-2, bcl-xL) that is followed by a late activation of pro apoptosis genes (bad, bak, bax) ALR is a liver growth factor and its serum levels atier 70% PH in rats peaks after 18 hours and return to normal after the 24th hour Moreover, ALR induces the expression of mitochondrial genes followed by an up-regulation of the oxidative phosphorylation ALR is mostly localised in the mitochondrial inter-membrane space and similarly to other factors in that site (Cytocrome C) can be considered a marker of mttochondrial integrity. One of the biological processes character&d by the alteration ofthe mitochondrial permeability is apoptosis Aim to define the effect of ALR on the expression of the apopiosis regulatory genes in the rat model of 70% hepatectomy. Materials and methods Ten male Fisher rats (F344) of 180-200 grams of body weight underwent 70% partial hepatectomy Five animals were treated i p with ALRp (0.5 w/day in saline) and five with albumin at 24. 36. 48 and 60 hours post surxery. The rats were &r&d at 48 and 72 hours post surgely We have analysed the iiver tissue for the activation of the anti-aooototic fbcl-2) and the ore-aooutotic ibax and ICE) genesbv RTPCR Results Our data show t&t at tie 72nd l& t&e is a dteady red&& ofth;proapoptotic genes mRNA (ICE -7O%, bax -50%) and an increased expression ofthe antiapoptotic gene mRNA (bcl-2 +30%) Conclusions. ALR modifies the expression of genes that regulate apoptosis induced by 70% PH in rats We argue that proliferatmn does not depend just on the growth factors but also on factors that inhibit cell death We believe that the release m the cytoplasm ofthe ALR is the biological process by which the cell contrasts apoptosis by preserving the maochondrial integrity in a phase of increased energy needs such during cell probferation
2P
F P RUSSO “, S TOMATO, M CONCONI §,P PARNlGOTTO 5, R NACCARATO BURRA”
‘, P
‘DIPARTIMENTO DI SClENZE CHIRURGICHE E GASTROENTEROLOGICHE; 8DlPARTIMENTO DI SCIENZE FARMACEUTICHE Introduction. Maintenance of different hepatocyte function in monolayer culture is generally short-lived Exogenous extracellular matrix (ECM) can mantain hepatocyte tin&n for a longer time. Whilst purified ECM components may be useful experimentally, there may be clinically bio-compatibility problems, such as clotting induced by collagen A biological support iniluencing cell development, migration, proliferation, shape and metabolic function would be very useful. The aim of the study was to obtain an homologous acellular matrix in order to support rat hepatocytes in primary culture. Materials and methods The HAM was prepared the day before each experiment by multiples rat liver slices. Slices were taken in sterile water, deoxycholic acid sodium salt solution and then digested with DNase. Experiments were performed on 25 male Sprague-Dawley rats Each animals was anesthetized The abdomen was opened and the liver was perfused in situ through vena porta with collagenase Isolated cells were counted in 1’ 10 diluted trypan blue and seededon 40 polystrene wells, 20 collagen coated wells and 60 HAM coated wells. Tbe number of viable cells was assayed by the thiawlyl blue (h4TT) assay based on mitochondrial reduction reaction Results About 8x106 of intact rat hepatocytes were obtained by each rat liver by using collagenasedigestion. Cell viability was about 80% The optimal seeding concentration was 2-3x105 cells/cm2 This concentration was used on polystyrene wells (chosen as control) and on collagen and HAM coated wells The MTT assays were performed at 24 hours and then daily after seeding Thanks to a calibration curve it was possible to express the absorbance value of MTT assay as number of viable cells (VC). 89 333, 50 000, 18.666 VC on polysyrene wells, 182.000, 101 333, 62 666 VC on collagen coated wells, 195 333, II2 666, 71 333 VC on HAM coated wells at 24, 96, 144 hours respectively The positivity ofthe MTT demonstrated a high activity of mitochondrium and the histology (emathoxylin-rosin and toluidin blu) showed a normal morphology ofhepatocytes cultured in the HAM Conclusion- These preliminary results suggest that hepatocytes in this new model of acellular biological matrix find their nomu. situation in terms of support and have at least an equal Sun&al as collagen
IP GENOM,C POLYMORPHISM nEALTHY lNDlvlDUALS
OF AUGMENTER OF LIVER REGENERATION
IN
M Margiona*, L Polimeno*, G Manco*. B Pesett~‘. E Ierardi#, R Francavdla’, S Morisco*, A Francavilla’
EXPRESSION OF HEAT SHOCK PROTEINS IN RESPONSE TO THE OXIDATIVE STRESS IN PATIENTS WITH CHRONIC LIVER DISEASES F Terrac&no*, A. Fed&x*, T De Sirnone’, C. Tuccillo*, E Fmamore”, C Loguerc~a*,C Del Vecchio Blanco, F Galdiero” ‘Gastroentemlogy School - 2nd University of Naples; ‘Institute ofMicrobiology - 2nd Unwersity of Naples
Introduction and Aim The genomic polymorphism, that is based on mutations ofDNA sequences, plays a key role both in the definition ofindividual phenotypic diversity and m disease ‘s predisposition The Augmenter of Liver Regeneration (ALR) gene. a well known hepatic growth factor, is located on the short arm ofchromosome 16 in humans and is composed of a sesuence of 2496 base pair It has been demonstrated that ALR play a crucial role in the mitochbndria bmgenesis DkA mutation of this factor may lead to functional mutation ofthe protein that may be of great imponance in diseases for which mitochondria metabolism is crucial (myastenic, astenospermic, neurological syndromes and hemocromatosis). At present little is known on the factors that control ALR gene expression and on the molecular pattern characteristic of the genomic polymorphism in healthy individuals therefore we have planned to explore this topic in our geographical area Material and methods. The genonuc DNA of 30 healthy indwlduals has been extracted from peripheric blood, amplified by nested PCR using specific pruners corresponding to two sequences of the second and third exon of the ALR gene The PCR products, less than 200 bp, have been separated by electrophoresis using the Smgle Strand Conformation Polvmomhism (SSCP) and silver-stained Usine this method, the aenomic sequences develor a c&nplex stm&e a&ding with their base &position with acharacter& electrophoretic pattern This method allows to recognise mutation extended to just a single base Results Our results, referred to the DNA sequences studied, have shown the electrophoretic pattern below reported In the healthy individuals of our geographic area the ALR genehave different allelic variations and the gene product keeps all the biological functions Conclusions. Our data, although preliminary on healthy individuals, represent for human the normal control to which refer for the ALR gene analysis in case of pathologies associated with mitochondrial metabolism dysfunction as yet not identitied
The family of Heat Shock Proteins HSP includes different forms on the basis of molecular weight These regulatory proteins are producted by cells of all organisms as response to stress. In human cells,HSP27 contribute to the stabilization of intracellular actin filaments and could play a regulatory role for the organization of the cytoskeleran Nitric oxlde~O),and particularly NO-thiol adducts(SH-NO),intluence the type and the entity of HSP expression Both HSP and NO affect genetranscription and apoptosis In experimental animals, HSP (60, 70, 90) were studied in relatmnship to cholestaas, as well as to NO and glutathione(GSH) production and limction. Several data documentedan increase of oxidative stress and a decreaseof GSH in patients with chronic liver damage We evaluated lymphocyte expression of HSP in relationship to SH-NO, glutathione-stransferase(GST) and markers of oxidative stress (malan,ldialdehyde.MA, and 4hydroxinonenal,4-HNE) plasma levels in patients with chronic liver disease PATIENTS- 16 with HCV-related biopsy-proven chronic hepatitis(CH), 16 with HCVrelated and 5 with alcoholic cirrhosis (10 Child A, I IChild B+C) Ten healthy subjects as control group METHODS HSP expression was evaluatedby Western blot, SH-NO by spectraphotometric method, GST, MDA and 4.HNE by commercial kits RESULTS HSP60 was positive m 5% of all patients globally cansldered and m none control HSPZ7,ahsentin controls, was expressed in 46,7% CH and 71.4% cmhotics (50% m Chdd A; 91% in Child B+C) SH-NO levels were increased in 88% of patients HSP27posrtive and in 30% of negative (mean *SD= 120 *23 vs 72 +25, p
Al37