Marcello Merola | Università degli Studi di Napoli "Federico II" (original) (raw)

Papers by Marcello Merola

AIDS Research and Human Retroviruses, 1996

Productive infection by the LAV strain has been demonstrated in T cell precursors at different st... more Productive infection by the LAV strain has been demonstrated in T cell precursors at different stages of intrathymic development, while viral replication in thymic epithelial cells is still controversial. In this article we show that epithelial cell cultures derived from the medullary component of normal thymus are infectable by HTLV-IIIB virus through cell-free and lymphoid-mediated transmission. Free virus inoculum results in the integration of proviral copies undergoing poor replication, whereas lymphoid-mediated transmission leads to substantial viral expression and the production of viral progeny able to secondary infect lymphoid cells. Interleukin 6 production and phenotype changes (increased expression of MHC class I and ICAM-1) were induced in TE cells by contact with free virus or by adhesion to infected lymphoid cells. By contrast, NF-kappa B-binding activity on the HIV-1 LTR kappa B enhancer element was upregulated only by contact with infected lymphoid cells, but not with virus. The viral replication observed in TE cells after lymphoid-mediated transmission correlates with the upregulation of NF-kappa B-binding activity. Interleukin 6 increased production and phenotype changes and increased NF-kappa B-binding activity were also induced by adhesion to uninfected lymphoid cells, demonstrating that lymphoepithelial cell contacts can activate TE cells. These results demonstrate that thymic epithelial cells are permissive to HIV infection and that viral replication in this cell lineage can be modulated by intracellular signals delivered by adhesive contacts.

European Journal of Immunology, 2003

Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithel... more Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor § 6 g 4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-‹ B and NF-IL6 transactivators. Here we show that § 6 g 4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, g 4 cross-linking activated p38 and extracellular signalregulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and g 4 integrin-induced production of IL-6 preventing NF-‹ B and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-‹ B activation. Overall, our results indicate that p38 MAPK and § 6 g 4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses.

European journal of biochemistry / FEBS, 1996

A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two brea... more A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two breast-carcinoma-derived cell lines has been performed. MDA-MB-231 cells produce constitutive detectable levels of both secreted IL-6 and mRNA which, as expected, are dramatically enhanced following induction by either IL-1 beta or tumor necrosis factor-alpha (TNF-alpha). The levels of both secreted IL-6 and IL-6 mRNA are significantly higher in response to IL-1 beta in spite of the fact that stimulation by TNF-alpha alone enhances the half life of IL-6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL-6 in these cells. In contrast, MCF-7 cells fail to produce detectable IL-6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF-kappa B, NFIL6 and NFIL6 beta, which have been described to be sufficient to activate the IL-6 gene in other cell systems, shows a similar pattern of expression in both MCF-7 and MDA...

Current opinion in virology, Jan 27, 2015

Although almost 15 years have passed since the birthdate of Reverse Vaccinology (RV), there are v... more Although almost 15 years have passed since the birthdate of Reverse Vaccinology (RV), there are very limited applications of this approach to viral vaccines discovery. Undeniably, RV presents a series of advantages as it can virtually identify all potential antigens coded by a genome, irrespective of their abundance, phase of expression and immunogenicity. Additionally, it can be applied to all pathogens, including those that cannot be grown in vitro. In this review we summarize the few examples of RV application to viruses, in particular the Herpesviridae, and report the advantage and limitations of this approach. Next we focus on the novel approaches and additional technologies to vaccine development including structure based approach (Structural Vaccinology [SV]), synthetic biology and some examples of their application in the development of viral vaccines.

Cellular microbiology, Jan 19, 2015

Translocation of the nasopharyngeal barrier by Neisseria meningitidis occurs via an intracellular... more Translocation of the nasopharyngeal barrier by Neisseria meningitidis occurs via an intracellular microtubule-dependent pathway and represents a crucial step in its pathogenesis. Despite this fact, the interaction of invasive meningococci with host subcellular compartments and the resulting impact on their organization and function have not been investigated. The influence of serogroup B strain MC58 on the host cells polarity and intracellular trafficking system was assessed by confocal microscopy visualization of different plasma membrane associated components (such as E-Cadherin, ZO-1, and Transferrin receptor) and evaluation of the transferrin uptake and recycling in infected Calu-3 monolayers. Additionally, the association of N. meningitidis with different endosomal compartments was evaluated through the concomitant staining of bacteria and markers specific for Rab11, Rab22a, Rab25 and Rab3, followed by confocal microscopy imaging. Subversion of the host cell architecture and in...

Molecular and cellular biochemistry, 1998

Lithostathine may play a physiological role in preventing the precipitation of excess calcium in ... more Lithostathine may play a physiological role in preventing the precipitation of excess calcium in the pancreatic juice. The hypothesis has been advanced that in chronic calcifying pancreatitis the abnormal biosynthesis of lithostathine might be the original defect to which genetic proneness to the disease may be ascribed. The aim of the present work was to study lithostathine messenger RNA expression in the pancreas of patients with different types of pancreatitis. Lithostathine and chymotrypsinogen mRNA were determined in surgical specimens obtained from the pancreases of the following subjects: (a) 13 patients with chronic alcoholic pancreatitis (84.6% calcified); (b) 4 patients with chronic hereditary pancreatitis (all calcified); (c) 6 patients with chronic obstructive pancreatitis (4 calcified); and (d) 27 subjects suffering from pancreatic cancer. Significantly lower concentrations of both mRNAs were found in the pancreases of chronic pancreatitis patients than in non-cancerous...

Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 1996

The human interleukin-6 (IL-6) promoter contains two regulatory elements, a kappa B enhancer and ... more The human interleukin-6 (IL-6) promoter contains two regulatory elements, a kappa B enhancer and a NFIL-6 (C/EBP beta) binding site, which have been reported to be essential for inducibility of the IL-6 gene. We show that the kappa B element alone is sufficient to confer inducibility on the IL-6 gene in cells treated with either IL-1 beta or TNF-alpha. Gel-retardation analysis of nuclear extracts from IL-1 beta or TNF-alpha-treated cells using specific antibodies has shown that at least five retarded complexes bind to the IL-6 kappa B element in addition to NF-kappa B. Furthermore, apart from p50 (NF-kappa B1) and p65 (RelA), no other members of the Rel family are present in these complexes. Comparative analysis with the kappa B enhancer of the immunoglobulin kappa chain gene shows that three of these complexes bind specifically to the IL-6 kappa B enhancer: a complex of p50/NFIL6, a p65 homodimer, and a non-Rel-related constitutive protein. Finally, transfection experiments, in whi...

International Journal of Hepatology, 2011

The Hepatitis C virus E1 and E2 envelope proteins are the major players in all events required fo... more The Hepatitis C virus E1 and E2 envelope proteins are the major players in all events required for virus entry into target cells. In addition, the recently developed HCV cell culture system has indicated that E1E2 heterodimer formation is a prerequisite for viral particle production. In this paper, we explored a new genetic approach to construct intergenotypic 2a/1b chimeras, maintaining the structural region of the infectious strain JFH1 and substituting the soluble portion of E1 and/or E2 proteins. This strategy provides useful information on the role of the surface-exposed domain of the envelope proteins in virus morphogenesis and allows comparative analysis of different HCV genotypes. We found that substituting the E2 protein ectodomain region abolishes the production of chimeric infectious particles. Our data indicate that the soluble part of the E2 protein is involved in a genotypespecific interplay with remaining viral proteins that affect the HCV assembly process.

The Italian journal of biochemistry

Poly(ADP-ribose)synthetase has been purified over 600-fold from HeLa cell nuclei. Upon electropho... more Poly(ADP-ribose)synthetase has been purified over 600-fold from HeLa cell nuclei. Upon electrophoresis on 9% polyacrylamide slab-gel in the presence of SDS, the purified enzyme resolved in two bands that showed similar apparent Mr values, 110,000 and 118,000. The aminoacid composition of the two components differed from compositions reported for the same enzyme from other sources. In accordance with other reports, the enzyme purified from HeLa cell nuclei exhibited an absolute dependence on DNA and its activity was modulated by changes in the histone concentration. Incubation of intact nuclei from HeLa cells with [14C]NAD resulted in the isolation of a poly(ADP-ribose)synthetase to which a definite radioactivity is bound. These results are taken as a further demonstration that the enzyme can be auto-ADP-ribosylated.

The Journal of biological chemistry, Jan 25, 1989

The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino... more The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine w...

A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two brea... more A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two breast-carcinoma-derived cell lines has been performed. MDA-MB-231 cells produce constitutive detectable levels of both secreted 1L-6 and mRNA which, as expected. are dramatically enhanced following induction by either IL-1β or tumor necrosis factor-α (TNF-α). The levels of both secreted IL-6 and IL-6 mRNA are significantly higher in response to IL-1β in spite of the fact that stimulation by TNF-α alone enhances the half life of IL-6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL-6 in these cells. In contrast, MCF-7 cells fail to produce detectable IL-6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF-κB, NFIL6 and NFIL6β, which have been described to be sufficient to activate the IL-6 gene in other cell systems, shows a similar pattern of expression in both MCF-7 and MDA-MB-231 cells. Furthermore, t...

PLoS ONE, 2014

Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in t... more Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.

PLoS ONE, 2012

The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all pr... more The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcc). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Liganddependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.

Virology, 2005

The recombinant E1E2 heterodimer of the hepatitis C virus is a candidate for a subunit vaccine. F... more The recombinant E1E2 heterodimer of the hepatitis C virus is a candidate for a subunit vaccine. Folding analysis of E1 and E2 glycoproteins, stably expressed in CHO cells, showed that E1 folding was faster and more efficient than E2. The oxidized DTT-resistant conformation of E1 was completed within 2 h post-synthesis, while E2 not only required up to 6 h but also generated non-native species. Calnexin was found to assist E1 folding, whereas no chaperone association was found with E2. The assembly of E1 and E2 was assessed by co-immunoprecipitation and sedimentation velocity analysis. We found that the formation of native E1E2 heterodimers paralleled E2 oxidation kinetics, suggesting that E2 completed its folding process after association with E1. Once formed, sedimentation of the native E1E2 heterodimers was consistent with the absence of additional associated factors. Taken together, our data strongly suggest that productive folding of the major HCV spike protein E2 is assisted by E1. D

European Journal of Biochemistry, 1996

PLoS ONE, 2011

Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a genera... more Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.

Nucleic Acids Research, 1999

Expression of the interleukin-6 (IL-6) gene is usually tightly controlled and may be induced in s... more Expression of the interleukin-6 (IL-6) gene is usually tightly controlled and may be induced in specific tissues only after treatment with appropriate stimuli. The molecular mechanisms responsible for IL-6 gene repression in specific tissues or cell lines remain poorly defined. In order to address this question we have studied two human breast carcinoma cell lines, MDA-MB-231, in which the IL-6 gene is expressed, and MCF-7, in which it is not. The promoter region of the IL-6 gene was analysed in both cell lines with reference to two different parameters: (i) DNase I hypersensitivity; (ii) the in vivo pattern of DNA-protein interactions. We show herein that the mechanism responsible for silencing IL-6 gene expression in MCF-7 cells most probably involves a modification of chromatin structure, as suggested by a decreased sensitivity of the IL-6 promoter to DNase I relative to the IL-6-expressing cell line MDA-MB-231. Moreover, we show that a 'closed' nucleosomal structure in MCF-7 cells does not inhibit the binding of nuclear proteins to IL-6 gene regulatory sequences in vivo. We suggest, therefore, that, in non-expressing cells, local chromatin remodelling at the proximal promoter is inhibited by negative regulators, as suggested by two specific hallmarks of nuclear factor binding that are not observed in expressing cells: an additional in vivo footprint spanning positions -135/-119 and an additional DNase I hypersensitive site far upstream, around position -1400. Furthermore, a specific factor binding in vitro to the -140/-116 region of the IL-6 promoter is found in MCF-7 cells.

Nucleic Acids Research, 1999

The nuclear protein CBF1 has been shown to function as an intermediate to target transcription fa... more The nuclear protein CBF1 has been shown to function as an intermediate to target transcription factors, such as the activated Notch receptor, to specific DNA sites. In this paper, we show that CBF1 from cell lines of different origin is able to bind to the κ κ κ κB site of the IL-6 promoter. By transfection analyses performed in HeLa cells, we demonstrate that overexpressed CBF1 acts as a negative regulator of IL-6 gene transcription and is unable to elicit Notch-dependent activation of this gene. Analyses of protein-DNA interactions indicate that the topology of the complex formed by CBF1 and the target DNA is subtly affected by sequences surrounding the recognition site. Furthermore, we show that CBF1 induces DNA bending. This finding suggests that CBF1 may influence IL-6 gene transcription by determining a specific conformation of the promoter region.

AIDS Research and Human Retroviruses, 1996

Productive infection by the LAV strain has been demonstrated in T cell precursors at different st... more Productive infection by the LAV strain has been demonstrated in T cell precursors at different stages of intrathymic development, while viral replication in thymic epithelial cells is still controversial. In this article we show that epithelial cell cultures derived from the medullary component of normal thymus are infectable by HTLV-IIIB virus through cell-free and lymphoid-mediated transmission. Free virus inoculum results in the integration of proviral copies undergoing poor replication, whereas lymphoid-mediated transmission leads to substantial viral expression and the production of viral progeny able to secondary infect lymphoid cells. Interleukin 6 production and phenotype changes (increased expression of MHC class I and ICAM-1) were induced in TE cells by contact with free virus or by adhesion to infected lymphoid cells. By contrast, NF-kappa B-binding activity on the HIV-1 LTR kappa B enhancer element was upregulated only by contact with infected lymphoid cells, but not with virus. The viral replication observed in TE cells after lymphoid-mediated transmission correlates with the upregulation of NF-kappa B-binding activity. Interleukin 6 increased production and phenotype changes and increased NF-kappa B-binding activity were also induced by adhesion to uninfected lymphoid cells, demonstrating that lymphoepithelial cell contacts can activate TE cells. These results demonstrate that thymic epithelial cells are permissive to HIV infection and that viral replication in this cell lineage can be modulated by intracellular signals delivered by adhesive contacts.

European Journal of Immunology, 2003

Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithel... more Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor § 6 g 4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-‹ B and NF-IL6 transactivators. Here we show that § 6 g 4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, g 4 cross-linking activated p38 and extracellular signalregulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and g 4 integrin-induced production of IL-6 preventing NF-‹ B and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-‹ B activation. Overall, our results indicate that p38 MAPK and § 6 g 4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses.

European journal of biochemistry / FEBS, 1996

A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two brea... more A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two breast-carcinoma-derived cell lines has been performed. MDA-MB-231 cells produce constitutive detectable levels of both secreted IL-6 and mRNA which, as expected, are dramatically enhanced following induction by either IL-1 beta or tumor necrosis factor-alpha (TNF-alpha). The levels of both secreted IL-6 and IL-6 mRNA are significantly higher in response to IL-1 beta in spite of the fact that stimulation by TNF-alpha alone enhances the half life of IL-6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL-6 in these cells. In contrast, MCF-7 cells fail to produce detectable IL-6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF-kappa B, NFIL6 and NFIL6 beta, which have been described to be sufficient to activate the IL-6 gene in other cell systems, shows a similar pattern of expression in both MCF-7 and MDA...

Current opinion in virology, Jan 27, 2015

Although almost 15 years have passed since the birthdate of Reverse Vaccinology (RV), there are v... more Although almost 15 years have passed since the birthdate of Reverse Vaccinology (RV), there are very limited applications of this approach to viral vaccines discovery. Undeniably, RV presents a series of advantages as it can virtually identify all potential antigens coded by a genome, irrespective of their abundance, phase of expression and immunogenicity. Additionally, it can be applied to all pathogens, including those that cannot be grown in vitro. In this review we summarize the few examples of RV application to viruses, in particular the Herpesviridae, and report the advantage and limitations of this approach. Next we focus on the novel approaches and additional technologies to vaccine development including structure based approach (Structural Vaccinology [SV]), synthetic biology and some examples of their application in the development of viral vaccines.

Cellular microbiology, Jan 19, 2015

Translocation of the nasopharyngeal barrier by Neisseria meningitidis occurs via an intracellular... more Translocation of the nasopharyngeal barrier by Neisseria meningitidis occurs via an intracellular microtubule-dependent pathway and represents a crucial step in its pathogenesis. Despite this fact, the interaction of invasive meningococci with host subcellular compartments and the resulting impact on their organization and function have not been investigated. The influence of serogroup B strain MC58 on the host cells polarity and intracellular trafficking system was assessed by confocal microscopy visualization of different plasma membrane associated components (such as E-Cadherin, ZO-1, and Transferrin receptor) and evaluation of the transferrin uptake and recycling in infected Calu-3 monolayers. Additionally, the association of N. meningitidis with different endosomal compartments was evaluated through the concomitant staining of bacteria and markers specific for Rab11, Rab22a, Rab25 and Rab3, followed by confocal microscopy imaging. Subversion of the host cell architecture and in...

Molecular and cellular biochemistry, 1998

Lithostathine may play a physiological role in preventing the precipitation of excess calcium in ... more Lithostathine may play a physiological role in preventing the precipitation of excess calcium in the pancreatic juice. The hypothesis has been advanced that in chronic calcifying pancreatitis the abnormal biosynthesis of lithostathine might be the original defect to which genetic proneness to the disease may be ascribed. The aim of the present work was to study lithostathine messenger RNA expression in the pancreas of patients with different types of pancreatitis. Lithostathine and chymotrypsinogen mRNA were determined in surgical specimens obtained from the pancreases of the following subjects: (a) 13 patients with chronic alcoholic pancreatitis (84.6% calcified); (b) 4 patients with chronic hereditary pancreatitis (all calcified); (c) 6 patients with chronic obstructive pancreatitis (4 calcified); and (d) 27 subjects suffering from pancreatic cancer. Significantly lower concentrations of both mRNAs were found in the pancreases of chronic pancreatitis patients than in non-cancerous...

Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 1996

The human interleukin-6 (IL-6) promoter contains two regulatory elements, a kappa B enhancer and ... more The human interleukin-6 (IL-6) promoter contains two regulatory elements, a kappa B enhancer and a NFIL-6 (C/EBP beta) binding site, which have been reported to be essential for inducibility of the IL-6 gene. We show that the kappa B element alone is sufficient to confer inducibility on the IL-6 gene in cells treated with either IL-1 beta or TNF-alpha. Gel-retardation analysis of nuclear extracts from IL-1 beta or TNF-alpha-treated cells using specific antibodies has shown that at least five retarded complexes bind to the IL-6 kappa B element in addition to NF-kappa B. Furthermore, apart from p50 (NF-kappa B1) and p65 (RelA), no other members of the Rel family are present in these complexes. Comparative analysis with the kappa B enhancer of the immunoglobulin kappa chain gene shows that three of these complexes bind specifically to the IL-6 kappa B enhancer: a complex of p50/NFIL6, a p65 homodimer, and a non-Rel-related constitutive protein. Finally, transfection experiments, in whi...

International Journal of Hepatology, 2011

The Hepatitis C virus E1 and E2 envelope proteins are the major players in all events required fo... more The Hepatitis C virus E1 and E2 envelope proteins are the major players in all events required for virus entry into target cells. In addition, the recently developed HCV cell culture system has indicated that E1E2 heterodimer formation is a prerequisite for viral particle production. In this paper, we explored a new genetic approach to construct intergenotypic 2a/1b chimeras, maintaining the structural region of the infectious strain JFH1 and substituting the soluble portion of E1 and/or E2 proteins. This strategy provides useful information on the role of the surface-exposed domain of the envelope proteins in virus morphogenesis and allows comparative analysis of different HCV genotypes. We found that substituting the E2 protein ectodomain region abolishes the production of chimeric infectious particles. Our data indicate that the soluble part of the E2 protein is involved in a genotypespecific interplay with remaining viral proteins that affect the HCV assembly process.

The Italian journal of biochemistry

Poly(ADP-ribose)synthetase has been purified over 600-fold from HeLa cell nuclei. Upon electropho... more Poly(ADP-ribose)synthetase has been purified over 600-fold from HeLa cell nuclei. Upon electrophoresis on 9% polyacrylamide slab-gel in the presence of SDS, the purified enzyme resolved in two bands that showed similar apparent Mr values, 110,000 and 118,000. The aminoacid composition of the two components differed from compositions reported for the same enzyme from other sources. In accordance with other reports, the enzyme purified from HeLa cell nuclei exhibited an absolute dependence on DNA and its activity was modulated by changes in the histone concentration. Incubation of intact nuclei from HeLa cells with [14C]NAD resulted in the isolation of a poly(ADP-ribose)synthetase to which a definite radioactivity is bound. These results are taken as a further demonstration that the enzyme can be auto-ADP-ribosylated.

The Journal of biological chemistry, Jan 25, 1989

The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino... more The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine w...

A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two brea... more A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two breast-carcinoma-derived cell lines has been performed. MDA-MB-231 cells produce constitutive detectable levels of both secreted 1L-6 and mRNA which, as expected. are dramatically enhanced following induction by either IL-1β or tumor necrosis factor-α (TNF-α). The levels of both secreted IL-6 and IL-6 mRNA are significantly higher in response to IL-1β in spite of the fact that stimulation by TNF-α alone enhances the half life of IL-6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL-6 in these cells. In contrast, MCF-7 cells fail to produce detectable IL-6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF-κB, NFIL6 and NFIL6β, which have been described to be sufficient to activate the IL-6 gene in other cell systems, shows a similar pattern of expression in both MCF-7 and MDA-MB-231 cells. Furthermore, t...

PLoS ONE, 2014

Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in t... more Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.

PLoS ONE, 2012

The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all pr... more The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcc). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Liganddependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.

Virology, 2005

The recombinant E1E2 heterodimer of the hepatitis C virus is a candidate for a subunit vaccine. F... more The recombinant E1E2 heterodimer of the hepatitis C virus is a candidate for a subunit vaccine. Folding analysis of E1 and E2 glycoproteins, stably expressed in CHO cells, showed that E1 folding was faster and more efficient than E2. The oxidized DTT-resistant conformation of E1 was completed within 2 h post-synthesis, while E2 not only required up to 6 h but also generated non-native species. Calnexin was found to assist E1 folding, whereas no chaperone association was found with E2. The assembly of E1 and E2 was assessed by co-immunoprecipitation and sedimentation velocity analysis. We found that the formation of native E1E2 heterodimers paralleled E2 oxidation kinetics, suggesting that E2 completed its folding process after association with E1. Once formed, sedimentation of the native E1E2 heterodimers was consistent with the absence of additional associated factors. Taken together, our data strongly suggest that productive folding of the major HCV spike protein E2 is assisted by E1. D

European Journal of Biochemistry, 1996

PLoS ONE, 2011

Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a genera... more Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.

Nucleic Acids Research, 1999

Expression of the interleukin-6 (IL-6) gene is usually tightly controlled and may be induced in s... more Expression of the interleukin-6 (IL-6) gene is usually tightly controlled and may be induced in specific tissues only after treatment with appropriate stimuli. The molecular mechanisms responsible for IL-6 gene repression in specific tissues or cell lines remain poorly defined. In order to address this question we have studied two human breast carcinoma cell lines, MDA-MB-231, in which the IL-6 gene is expressed, and MCF-7, in which it is not. The promoter region of the IL-6 gene was analysed in both cell lines with reference to two different parameters: (i) DNase I hypersensitivity; (ii) the in vivo pattern of DNA-protein interactions. We show herein that the mechanism responsible for silencing IL-6 gene expression in MCF-7 cells most probably involves a modification of chromatin structure, as suggested by a decreased sensitivity of the IL-6 promoter to DNase I relative to the IL-6-expressing cell line MDA-MB-231. Moreover, we show that a 'closed' nucleosomal structure in MCF-7 cells does not inhibit the binding of nuclear proteins to IL-6 gene regulatory sequences in vivo. We suggest, therefore, that, in non-expressing cells, local chromatin remodelling at the proximal promoter is inhibited by negative regulators, as suggested by two specific hallmarks of nuclear factor binding that are not observed in expressing cells: an additional in vivo footprint spanning positions -135/-119 and an additional DNase I hypersensitive site far upstream, around position -1400. Furthermore, a specific factor binding in vitro to the -140/-116 region of the IL-6 promoter is found in MCF-7 cells.

Nucleic Acids Research, 1999

The nuclear protein CBF1 has been shown to function as an intermediate to target transcription fa... more The nuclear protein CBF1 has been shown to function as an intermediate to target transcription factors, such as the activated Notch receptor, to specific DNA sites. In this paper, we show that CBF1 from cell lines of different origin is able to bind to the κ κ κ κB site of the IL-6 promoter. By transfection analyses performed in HeLa cells, we demonstrate that overexpressed CBF1 acts as a negative regulator of IL-6 gene transcription and is unable to elicit Notch-dependent activation of this gene. Analyses of protein-DNA interactions indicate that the topology of the complex formed by CBF1 and the target DNA is subtly affected by sequences surrounding the recognition site. Furthermore, we show that CBF1 induces DNA bending. This finding suggests that CBF1 may influence IL-6 gene transcription by determining a specific conformation of the promoter region.