Antonio Gaballo - Academia.edu (original) (raw)

Papers by Antonio Gaballo

Research paper thumbnail of Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leuckocytes from AIDS patients with human cytomegalovirus viraemia

Molecular and Cellular Probes, 1991

FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially H... more FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, whil~ 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMVrelated clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10 s cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCRpositive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.

Research paper thumbnail of Antibiotic Sensitivity of Bacillus clausii Strains in Commercial Preparation

Clinical Immunology, Endocrine & Metabolic Drugs, 2015

Recently has been acknowledged the healthy use of Bacillus and related bacteria as probiotics. A ... more Recently has been acknowledged the healthy use of Bacillus and related bacteria as probiotics. A mixture reported to contain four probiotic strains of Bacillus clausii is marketed as an OTC (Over The Counter) medicinal supplement for human use. Their poliantibiotic resistant property, useful for restoring the gut microbiota during antibiotic treatment, raises the question about the risk of resistance transfer. In order to better assess the risk-benefit ratio it is important to always monitoring the pattern and stability of resistance spectra in these bacteria. In this work, we have extensively redefined the antibiotic susceptibility profile of these four probiotic strains. Resistance phenotype has been determined by screening a large number of antibiotics, including natural products (such as penicillin, vancomycin and erythromycin), and completely synthetic molecules (such as fluoroquinolones). Extensive comparison with a wild type strain belonging to the normal intestinal microbiome was carried out. The molecular basis of some resistances was determined. Observed antibiotic resistances were correlated with previous and new data in safety evaluations of these strains for human use.

Research paper thumbnail of 1.6 The Mitochondrial F1Fo ATP Synthase

Handbook of Neurochemistry and Molecular Neurobiology, 2007

Research paper thumbnail of Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leuckocytes from AIDS patients with human cytomegalovirus viraemia

Molecular and Cellular Probes, 1991

FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially H... more FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, whil~ 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMVrelated clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10 s cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCRpositive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.

Research paper thumbnail of Inhibitory and Anchoring Domains in the ATPase Inhibitor Protein IF 1 of Bovine Heart Mitochondrial ATP Synthase

Journal of Bioenergetics and Biomembranes, 2000

The inhibitor protein IF 1 is a basic protein of 84 residues which inhibits the ATPase activity o... more The inhibitor protein IF 1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial F o F 1 -ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact F o F 1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF 1 (residues 42-58) binds to the α and β subunits of F 1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF 1 binds, simultaneously, to the OSCP subunit of F o in a pH-independent process. This binding keeps IF 1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.

Research paper thumbnail of Functional domains of the ATPase inhibitor protein from bovine heart mitochondria

FEBS Letters, 2000

A study is presented of the activity and temperature dependence of the ATPase inhibitor protein (... more A study is presented of the activity and temperature dependence of the ATPase inhibitor protein (IF 1 ) from bovine heart mitochondria and of synthetic partial IF 1 peptides. The results show that the IF 1 -(42^58) peptide is the most potent inhibitory domain of IF 1 . ß

Research paper thumbnail of Topological and Functional Relationship of Subunits F 1 -γ and F o I-PVP(b) in the Mitochondrial H + -ATP Synthase †

Biochemistry, 1998

Diamide treatment of the F0F1-ATP synthase in "inside out" submitochondrial par... more Diamide treatment of the F0F1-ATP synthase in "inside out" submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated Fo reconstituted with F1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the F0I-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F1-gamma, both located in the stalk. The F0I-PVP(b) and F1-gamma cross-linking cause dramatic enhancement of oligomycin-sensitive decay of Delta mu H+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that F0I-PVP(b) and F1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of F0I-PVP(b) and the short alpha-helix of F1-gamma can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated F0, diamide also induces cross-linking of OSCP with another subunit of F0, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of Delta mu H+ generated by respiration, neither cross-linking between F0I-PVP(b) and F1-gamma subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by Delta mu H+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by Delta mu H+ decay through Fo induces a relative motion and/or a separation of the F0I-PVP(b) subunit and F1-gamma which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.

Research paper thumbnail of Cytoskeletal alterations of parkin-mutant human primary fibroblasts

Journal of Biotechnology, 2014

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have b... more Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Research paper thumbnail of Identification of a Meningococcal L-Glutamate ABC Transporter Operon Essential for Growth in Low-Sodium Environments

Infection and Immunity, 2006

GdhR is a meningococcal transcriptional regulator that was previously shown to positively control... more GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific L-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in L-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of L-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing L-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the L-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.

Research paper thumbnail of Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson’s disease patients

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2015

Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp... more Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, proteins and RNA processing.

Research paper thumbnail of An active mitochondrial biogenesis occurs during dendritic cell differentiation

The international journal of biochemistry & cell biology, 2012

Dendritic cells (DC) are sentinels of the immune system deriving from circulating monocyte precur... more Dendritic cells (DC) are sentinels of the immune system deriving from circulating monocyte precursors recruited to sites of inflammation. In a previous report (Del Prete et al., 2008) we showed that, after differentiation, DC exhibited increased number of condensed mitochondria and dynamic changes in their energy metabolism. A study is presented here showing that the DC differentiation process is characterized by increased expression level and activity of mitochondrial respiratory complexes, as well as by an increased mitochondrial DNA (mtDNA) copy number. Moreover, DC are equipped with more efficient antioxidant protection systems, over expressed most likely to detoxify increased ROS production, as a consequence of the much higher mitochondrial activity. Kinetic analysis of the three main mitochondrial biogenesis-associated genes revealed that the peak in PPARγ coactivator-1alpha (PGC-1α) gene expression was suddenly reached few hours after the onset of the differentiation. While P...

Research paper thumbnail of The structural and functional connection between the catalytic and proton translocating sectors of the mitochondrial F1F0-ATP synthase

The structural and functional connection between the peripheral catalytic F1 sector and the proto... more The structural and functional connection between the peripheral catalytic F1 sector and the proton-translocating membrane sector F0 of the mitochondrial ATP synthase is reviewed. The observations examined show that the N-terminus of subunit gamma, the carboxy-terminal and central region of F0I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateral stalk, which connects the peripheries of F1 to F0 and surrounds the central element of the stalk, constituted by subunits gamma and delta. The ATPase inhibitor protein (IF1) binds at one side of the F1F0 connection. The carboxy-terminal segment of IF1 apparently binds to OSCP. The 42L-58K segment of IF1, which is per se the most active domain of the protein, binds at the surface of one of the three alpha/beta pairs of F1, thus preventing the cyclic interconversion of the catalytic sites required for ATP hydrolysis.

Research paper thumbnail of Cytoskeletal Alterations and Biomechanical Properties of parkin-Mutant Human Primary Fibroblasts

Cell Biochemistry and Biophysics, 2014

Research paper thumbnail of Cytoskeletal alterations of parkin-mutant human primary fibroblasts

Journal of Biotechnology, 2014

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have b... more Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Research paper thumbnail of The Oxidative Phosphorylation System in Mammalian Mitochondria

Advances in Experimental Medicine and Biology, 2011

The chapter provides a review of the state of art of the oxidative phosphorylation system in mamm... more The chapter provides a review of the state of art of the oxidative phosphorylation system in mammalian mitochondria. The sections of the paper deal with:

Research paper thumbnail of Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease

Biochimica et biophysica acta, 2014

Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the mol... more Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activate...

Research paper thumbnail of F1 and F0 connections in the bovine mitochondrial ATP synthase

European Journal of Biochemistry, 2000

We have studied the functional effect of limited proteolysis by trypsin of the constituent subuni... more We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F 1 F 0 complex and isolated F 1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated.

Research paper thumbnail of Structure and expression of the atp operon coding for F1F0-ATP synthase from the antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727

Research in Microbiology, 2006

Nonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the glycopeptide an... more Nonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the glycopeptide antibiotic A40926. In this study, the nucleotide sequence of the atp operon coding for F 1 F 0 -ATP synthase of Nonomuraea sp. ATCC 39727 was determined. It consisted of ten open reading frames arranged in the order atpI (encoding the i protein), orfX, atpB (a subunit), atpE (c subunit), atpF (b subunit), atpH (δ subunit), atpA (α subunit), atpG (γ subunit), atpD (β subunit) and atpC (ε subunit). The orfX coded for a putative small hydrophobic 71 amino acid peptide of unknown function related to several bacterial permeases. Its presence appeared to be a distinctive feature of the atp operon of phylogenetically distant actinobacteria. Transcription of the atp operon was evaluated. The results of northern blot and RT-PCR experiments demonstrated that the atp genes were co-transcribed into a single polycistronic mRNA. Real-time RT-PCR data provided evidence showing that transcription of the atp operon was biphasic during Nonomuraea growth. The amount of the atpD transcript decreased at the end of the exponential growth phase, and then moderately increased during the early stationary phase when, in contrast, the levels of ctaC, encoding the cytochrome c oxidase subunit II, progressively decreased. Western blot analysis confirmed that ATP synthase was also present in the membrane during the stationary phase. These results together with previous data demonstrate that oligomycin-sensitive ATP-driven proton pumping activity remained constant in the stationary phase; in contrast, the activity and cytochrome content of the respiratory enzymes became negligible.

Research paper thumbnail of Migrainous infarction: association with vascular risk factors in a male subject

Neurological Sciences, 2009

Migraine with aura (MA) is associated with an increased risk of ischemic stroke, especially in yo... more Migraine with aura (MA) is associated with an increased risk of ischemic stroke, especially in young women with vascular risk factors (smoke, contraceptive pill). Patent foramen ovale (PFO) has also been associated with MA. We describe a 41-year-old man, in good health, with MA since 16, familiar history of diabetes, heavy smoker (30 cigarettes/day). Frequency (1-2 attacks/year) and clinical features of migraine have been unchanged since the onset. A few days before our examination he suffered a typical migraine attack. In the following hours, however, the headache became more and more throbbing and the aura symptoms (regressed as usual in 30 min) reappeared and persisted, so he went to an Emergency Department. The CT-scan (without contrast) was normal. The following days he had visual disturbances and spatial disorientation. We found a normal neurological examination and fundus oculi. He referred persisting visual troubles. We prescribed MR + angioMR which confirmed a migrainous infarction and ruled out others pathological conditions. Further tests found out dyslipidemia, hyperhomocysteinemia, impaired glucose tolerance. Transcranial Doppler showed right to left shunting. We also prescribed the screening tests for vasculitis (normal). In our opinion this case highlights the relevance of vascular risk factors in MA complications also in male subjects.

Research paper thumbnail of Comparative proteomic analysis of four Bacillus clausii strains: Proteomic expression signature distinguishes protein profile of the strains

Journal of Proteomics, 2011

A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry... more A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth. Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT-PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains.

Research paper thumbnail of Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leuckocytes from AIDS patients with human cytomegalovirus viraemia

Molecular and Cellular Probes, 1991

FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially H... more FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, whil~ 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMVrelated clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10 s cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCRpositive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.

Research paper thumbnail of Antibiotic Sensitivity of Bacillus clausii Strains in Commercial Preparation

Clinical Immunology, Endocrine & Metabolic Drugs, 2015

Recently has been acknowledged the healthy use of Bacillus and related bacteria as probiotics. A ... more Recently has been acknowledged the healthy use of Bacillus and related bacteria as probiotics. A mixture reported to contain four probiotic strains of Bacillus clausii is marketed as an OTC (Over The Counter) medicinal supplement for human use. Their poliantibiotic resistant property, useful for restoring the gut microbiota during antibiotic treatment, raises the question about the risk of resistance transfer. In order to better assess the risk-benefit ratio it is important to always monitoring the pattern and stability of resistance spectra in these bacteria. In this work, we have extensively redefined the antibiotic susceptibility profile of these four probiotic strains. Resistance phenotype has been determined by screening a large number of antibiotics, including natural products (such as penicillin, vancomycin and erythromycin), and completely synthetic molecules (such as fluoroquinolones). Extensive comparison with a wild type strain belonging to the normal intestinal microbiome was carried out. The molecular basis of some resistances was determined. Observed antibiotic resistances were correlated with previous and new data in safety evaluations of these strains for human use.

Research paper thumbnail of 1.6 The Mitochondrial F1Fo ATP Synthase

Handbook of Neurochemistry and Molecular Neurobiology, 2007

Research paper thumbnail of Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leuckocytes from AIDS patients with human cytomegalovirus viraemia

Molecular and Cellular Probes, 1991

FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially H... more FiftyAIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, whil~ 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMVrelated clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10 s cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCRpositive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.

Research paper thumbnail of Inhibitory and Anchoring Domains in the ATPase Inhibitor Protein IF 1 of Bovine Heart Mitochondrial ATP Synthase

Journal of Bioenergetics and Biomembranes, 2000

The inhibitor protein IF 1 is a basic protein of 84 residues which inhibits the ATPase activity o... more The inhibitor protein IF 1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial F o F 1 -ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact F o F 1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF 1 (residues 42-58) binds to the α and β subunits of F 1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF 1 binds, simultaneously, to the OSCP subunit of F o in a pH-independent process. This binding keeps IF 1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.

Research paper thumbnail of Functional domains of the ATPase inhibitor protein from bovine heart mitochondria

FEBS Letters, 2000

A study is presented of the activity and temperature dependence of the ATPase inhibitor protein (... more A study is presented of the activity and temperature dependence of the ATPase inhibitor protein (IF 1 ) from bovine heart mitochondria and of synthetic partial IF 1 peptides. The results show that the IF 1 -(42^58) peptide is the most potent inhibitory domain of IF 1 . ß

Research paper thumbnail of Topological and Functional Relationship of Subunits F 1 -γ and F o I-PVP(b) in the Mitochondrial H + -ATP Synthase †

Biochemistry, 1998

Diamide treatment of the F0F1-ATP synthase in &quot;inside out&quot; submitochondrial par... more Diamide treatment of the F0F1-ATP synthase in &quot;inside out&quot; submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated Fo reconstituted with F1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the F0I-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F1-gamma, both located in the stalk. The F0I-PVP(b) and F1-gamma cross-linking cause dramatic enhancement of oligomycin-sensitive decay of Delta mu H+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that F0I-PVP(b) and F1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of F0I-PVP(b) and the short alpha-helix of F1-gamma can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated F0, diamide also induces cross-linking of OSCP with another subunit of F0, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of Delta mu H+ generated by respiration, neither cross-linking between F0I-PVP(b) and F1-gamma subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by Delta mu H+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by Delta mu H+ decay through Fo induces a relative motion and/or a separation of the F0I-PVP(b) subunit and F1-gamma which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.

Research paper thumbnail of Cytoskeletal alterations of parkin-mutant human primary fibroblasts

Journal of Biotechnology, 2014

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have b... more Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Research paper thumbnail of Identification of a Meningococcal L-Glutamate ABC Transporter Operon Essential for Growth in Low-Sodium Environments

Infection and Immunity, 2006

GdhR is a meningococcal transcriptional regulator that was previously shown to positively control... more GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific L-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in L-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of L-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing L-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the L-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.

Research paper thumbnail of Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson’s disease patients

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2015

Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp... more Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, proteins and RNA processing.

Research paper thumbnail of An active mitochondrial biogenesis occurs during dendritic cell differentiation

The international journal of biochemistry & cell biology, 2012

Dendritic cells (DC) are sentinels of the immune system deriving from circulating monocyte precur... more Dendritic cells (DC) are sentinels of the immune system deriving from circulating monocyte precursors recruited to sites of inflammation. In a previous report (Del Prete et al., 2008) we showed that, after differentiation, DC exhibited increased number of condensed mitochondria and dynamic changes in their energy metabolism. A study is presented here showing that the DC differentiation process is characterized by increased expression level and activity of mitochondrial respiratory complexes, as well as by an increased mitochondrial DNA (mtDNA) copy number. Moreover, DC are equipped with more efficient antioxidant protection systems, over expressed most likely to detoxify increased ROS production, as a consequence of the much higher mitochondrial activity. Kinetic analysis of the three main mitochondrial biogenesis-associated genes revealed that the peak in PPARγ coactivator-1alpha (PGC-1α) gene expression was suddenly reached few hours after the onset of the differentiation. While P...

Research paper thumbnail of The structural and functional connection between the catalytic and proton translocating sectors of the mitochondrial F1F0-ATP synthase

The structural and functional connection between the peripheral catalytic F1 sector and the proto... more The structural and functional connection between the peripheral catalytic F1 sector and the proton-translocating membrane sector F0 of the mitochondrial ATP synthase is reviewed. The observations examined show that the N-terminus of subunit gamma, the carboxy-terminal and central region of F0I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateral stalk, which connects the peripheries of F1 to F0 and surrounds the central element of the stalk, constituted by subunits gamma and delta. The ATPase inhibitor protein (IF1) binds at one side of the F1F0 connection. The carboxy-terminal segment of IF1 apparently binds to OSCP. The 42L-58K segment of IF1, which is per se the most active domain of the protein, binds at the surface of one of the three alpha/beta pairs of F1, thus preventing the cyclic interconversion of the catalytic sites required for ATP hydrolysis.

Research paper thumbnail of Cytoskeletal Alterations and Biomechanical Properties of parkin-Mutant Human Primary Fibroblasts

Cell Biochemistry and Biophysics, 2014

Research paper thumbnail of Cytoskeletal alterations of parkin-mutant human primary fibroblasts

Journal of Biotechnology, 2014

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have b... more Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Research paper thumbnail of The Oxidative Phosphorylation System in Mammalian Mitochondria

Advances in Experimental Medicine and Biology, 2011

The chapter provides a review of the state of art of the oxidative phosphorylation system in mamm... more The chapter provides a review of the state of art of the oxidative phosphorylation system in mammalian mitochondria. The sections of the paper deal with:

Research paper thumbnail of Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease

Biochimica et biophysica acta, 2014

Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the mol... more Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activate...

Research paper thumbnail of F1 and F0 connections in the bovine mitochondrial ATP synthase

European Journal of Biochemistry, 2000

We have studied the functional effect of limited proteolysis by trypsin of the constituent subuni... more We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F 1 F 0 complex and isolated F 1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated.

Research paper thumbnail of Structure and expression of the atp operon coding for F1F0-ATP synthase from the antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727

Research in Microbiology, 2006

Nonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the glycopeptide an... more Nonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the glycopeptide antibiotic A40926. In this study, the nucleotide sequence of the atp operon coding for F 1 F 0 -ATP synthase of Nonomuraea sp. ATCC 39727 was determined. It consisted of ten open reading frames arranged in the order atpI (encoding the i protein), orfX, atpB (a subunit), atpE (c subunit), atpF (b subunit), atpH (δ subunit), atpA (α subunit), atpG (γ subunit), atpD (β subunit) and atpC (ε subunit). The orfX coded for a putative small hydrophobic 71 amino acid peptide of unknown function related to several bacterial permeases. Its presence appeared to be a distinctive feature of the atp operon of phylogenetically distant actinobacteria. Transcription of the atp operon was evaluated. The results of northern blot and RT-PCR experiments demonstrated that the atp genes were co-transcribed into a single polycistronic mRNA. Real-time RT-PCR data provided evidence showing that transcription of the atp operon was biphasic during Nonomuraea growth. The amount of the atpD transcript decreased at the end of the exponential growth phase, and then moderately increased during the early stationary phase when, in contrast, the levels of ctaC, encoding the cytochrome c oxidase subunit II, progressively decreased. Western blot analysis confirmed that ATP synthase was also present in the membrane during the stationary phase. These results together with previous data demonstrate that oligomycin-sensitive ATP-driven proton pumping activity remained constant in the stationary phase; in contrast, the activity and cytochrome content of the respiratory enzymes became negligible.

Research paper thumbnail of Migrainous infarction: association with vascular risk factors in a male subject

Neurological Sciences, 2009

Migraine with aura (MA) is associated with an increased risk of ischemic stroke, especially in yo... more Migraine with aura (MA) is associated with an increased risk of ischemic stroke, especially in young women with vascular risk factors (smoke, contraceptive pill). Patent foramen ovale (PFO) has also been associated with MA. We describe a 41-year-old man, in good health, with MA since 16, familiar history of diabetes, heavy smoker (30 cigarettes/day). Frequency (1-2 attacks/year) and clinical features of migraine have been unchanged since the onset. A few days before our examination he suffered a typical migraine attack. In the following hours, however, the headache became more and more throbbing and the aura symptoms (regressed as usual in 30 min) reappeared and persisted, so he went to an Emergency Department. The CT-scan (without contrast) was normal. The following days he had visual disturbances and spatial disorientation. We found a normal neurological examination and fundus oculi. He referred persisting visual troubles. We prescribed MR + angioMR which confirmed a migrainous infarction and ruled out others pathological conditions. Further tests found out dyslipidemia, hyperhomocysteinemia, impaired glucose tolerance. Transcranial Doppler showed right to left shunting. We also prescribed the screening tests for vasculitis (normal). In our opinion this case highlights the relevance of vascular risk factors in MA complications also in male subjects.

Research paper thumbnail of Comparative proteomic analysis of four Bacillus clausii strains: Proteomic expression signature distinguishes protein profile of the strains

Journal of Proteomics, 2011

A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry... more A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth. Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT-PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains.