A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites ... more A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites on the nicotinic acid pathway. Kynurenine, tryptophan, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid and xanthurenic acid were isocratically eluted and completely resolved with a mobile phase of acetonitrile + sodium acetate buffer, pH 4.76 (4:96, v/v). The flow rate was 0.8 mL/min at the beginning and was then linearly increased to 1.2 after 6 min; after 14 min the flow was augmented from 1.2 to 2 mL/min. The effluent was monitored with a variable UV detector set at 254 nm for the first five peaks and at 280 and 325 nm for the penultimate peak and final peak. Analytical recoveries of the compounds after deproteinization varied between 64% and 98%. The reported method should enable one to examine easily, extensively, quantitatively and routinely urinary tryptophan and the most important metabolites of the nicotinic acid pathway.
A sensitive and selective high performance liquid chromatographic doxophylline assay with ultravi... more A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.
Abstract: Previous investigations demonstrated that melatonin exerts an oncostatic action on est... more Abstract: Previous investigations demonstrated that melatonin exerts an oncostatic action on estrogen-responsive breast cancer, both in vitro and in vivo. Nevertheless, the pro-apoptotic effect of melatonin is still a matter of debate. An experimental study was undertaken to focus on melatonin-related apoptosis and to identify the apoptotic pathways involved. Whole cell-count, flow-cytometry analysis and proteins involved in apoptotic pathways [p53, p73, murine double minute 2 (MDM2), caspases-9,-7,-6, cleaved-poly ADP ribose polymerase (PARP), Bcl-2, Bax and apoptotic inducing factor (AIF)] were investigated in human MCF-7 breast cancer cells treated with physiological (1 nM) concentration of melatonin. Melatonin exerts a significant growth-inhibitory effect on MCF-7 cells, becoming evident after 72 hr and thereafter increasing linearly up to 144 hr. In this model, the growth-inhibition is transforming growth factor beta 1 (TGFβ1)-dependent and it might be reversed by adding an anti-TGFβ1 antibody. Melatonin induces a significant rise in apoptotic rate, at both 24 and 96 hr. The anti-TGFβ1 antibody almost completely suppresses melatonin-related late apoptosis; however, early apoptosis is unaffected. Early programmed cell death is associated with a significant increase in the p53/MDM2 ratio and in AIF release, without modifications in caspase activity or cleaved-PARP levels. Activated caspases-9 and -7 and cleaved-PARP increased significantly at 96 hr, concomitantly with a down-regulation of the Bcl-2/Bax ratio. These data suggest that two distinct apoptotic processes are triggered by melatonin in MCF-7 cells: an early, TGFβ1 and caspase-independent response, and a late apoptotic TGFβ1-dependent process in which activated-caspase-7 is likely to be the terminal effector.
In the past decades, an enormous amount of precious information has been collected about molecula... more In the past decades, an enormous amount of precious information has been collected about molecular and genetic characteristics of cancer. This knowledge is mainly based on a reductionistic approach, meanwhile cancer is widely recognized to be a ‘system biology disease’. The behavior of complex physiological processes cannot be understood simply by knowing how the parts work in isolation. There is not solely a matter how to integrate all available knowledge in such a way that we can still deal with complexity, but we must be aware that a deeply transformation of the currently accepted oncologic paradigm is urgently needed. We have to think in terms of biological networks: understanding of complex functions may in fact be impossible without taking into consideration influences (rules and constraints) outside of the genome. Systems Biology involves connecting experimental unsupervised multivariate data to mathematical and computational approach than can simulate biologic systems for hypothesis testing or that can account for what it is not known from high-throughput data sets. Metabolomics could establish the requested link between genotype and phenotype, providing informations that ensure an integrated understanding of pathogenic mechanisms and metabolic phenotypes and provide a screening tool for new targeted drug.
A rapid resolution liquid chromatography-tandem mass spectrometry analysis was applied to human u... more A rapid resolution liquid chromatography-tandem mass spectrometry analysis was applied to human urine samples to discover new molecular biomarkers of the alteration of psychophysical well-being due to the environmental, physiological and confinement stress conditions achieved in the MARS500 project. Urine samples of crew members were submitted to metabolomic studies by means of separative techniques coupled with mass spectrometric techniques and data analysis. More than 2,000 metabolite features were found in each comparison, and more than 150 metabolites were identified for every differential analysis. Significant modifications in eight metabolites were shared by the subjects under study.
Vestibular schwannoma (VS) often displays an irregular growth pattern due to factors which remain... more Vestibular schwannoma (VS) often displays an irregular growth pattern due to factors which remain unknown. In this study, VS cell lines from tissue removed during surgery were cultured in order to assess the presence of transforming growth factor beta-1 (TGFbeta-1) a multifunctional polypeptide that has different pleiotropic effects on various tissues. The antibody-binding inhibition assay technique was used. The proliferation rate of VS cell lines in vitro was assessed by [3H]thymidine incorporation, with determinations carried out on days 0, 1, 2 and 4 after removal of fetal calf serum (FCS) from the medium. After 48 h of FCS deprivation, cell growth decreased. TGFbeta-1 release also decreased progressively 24 h after removing FCS from the medium, reaching an overall decrease of 50% after 3 days. The cell culturing procedure appeared suitable for VS cells, in that VS cultures were viable for at least 4 months. TGFbeta-1 was initially shown to be released in significant amounts, but also to decrease progressively to values five times lower after 4 days. These data thus support the possibility of a significant association between TGFbeta-1 release and VS cell replication.
A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization ti... more A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87–96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g−1 and 0.15 mg g−1, respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation.
Background.Evidence has accumulated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is involved in t... more Background.Evidence has accumulated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is involved in the regulation of the proliferation of breast tumor cells. For complete tumor suppression high hypercalcemic doses of 1,25-(OH)2D3 are needed. The aim of this study was to assess the effect of combined treatment of 1,25-(OH)2D3 at low doses and melatonin (MEL) on the proliferation of estrogen-responsive rat breast cancer cell
A sensitive and selective high performance liquid chromatographic doxophylline assay with ultravi... more A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.
A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites ... more A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites on the nicotinic acid pathway. Kynurenine, tryptophan, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid and xanthurenic acid were isocratically eluted and completely resolved with a mobile phase of acetonitrile + sodium acetate buffer, pH 4.76 (4:96, v/v). The flow rate was 0.8 mL/min at the beginning and was then linearly increased to 1.2 after 6 min; after 14 min the flow was augmented from 1.2 to 2 mL/min. The effluent was monitored with a variable UV detector set at 254 nm for the first five peaks and at 280 and 325 nm for the penultimate peak and final peak. Analytical recoveries of the compounds after deproteinization varied between 64% and 98%. The reported method should enable one to examine easily, extensively, quantitatively and routinely urinary tryptophan and the most important metabolites of the nicotinic acid pathway.
A sensitive and selective high performance liquid chromatographic doxophylline assay with ultravi... more A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.
Abstract: Previous investigations demonstrated that melatonin exerts an oncostatic action on est... more Abstract: Previous investigations demonstrated that melatonin exerts an oncostatic action on estrogen-responsive breast cancer, both in vitro and in vivo. Nevertheless, the pro-apoptotic effect of melatonin is still a matter of debate. An experimental study was undertaken to focus on melatonin-related apoptosis and to identify the apoptotic pathways involved. Whole cell-count, flow-cytometry analysis and proteins involved in apoptotic pathways [p53, p73, murine double minute 2 (MDM2), caspases-9,-7,-6, cleaved-poly ADP ribose polymerase (PARP), Bcl-2, Bax and apoptotic inducing factor (AIF)] were investigated in human MCF-7 breast cancer cells treated with physiological (1 nM) concentration of melatonin. Melatonin exerts a significant growth-inhibitory effect on MCF-7 cells, becoming evident after 72 hr and thereafter increasing linearly up to 144 hr. In this model, the growth-inhibition is transforming growth factor beta 1 (TGFβ1)-dependent and it might be reversed by adding an anti-TGFβ1 antibody. Melatonin induces a significant rise in apoptotic rate, at both 24 and 96 hr. The anti-TGFβ1 antibody almost completely suppresses melatonin-related late apoptosis; however, early apoptosis is unaffected. Early programmed cell death is associated with a significant increase in the p53/MDM2 ratio and in AIF release, without modifications in caspase activity or cleaved-PARP levels. Activated caspases-9 and -7 and cleaved-PARP increased significantly at 96 hr, concomitantly with a down-regulation of the Bcl-2/Bax ratio. These data suggest that two distinct apoptotic processes are triggered by melatonin in MCF-7 cells: an early, TGFβ1 and caspase-independent response, and a late apoptotic TGFβ1-dependent process in which activated-caspase-7 is likely to be the terminal effector.
In the past decades, an enormous amount of precious information has been collected about molecula... more In the past decades, an enormous amount of precious information has been collected about molecular and genetic characteristics of cancer. This knowledge is mainly based on a reductionistic approach, meanwhile cancer is widely recognized to be a ‘system biology disease’. The behavior of complex physiological processes cannot be understood simply by knowing how the parts work in isolation. There is not solely a matter how to integrate all available knowledge in such a way that we can still deal with complexity, but we must be aware that a deeply transformation of the currently accepted oncologic paradigm is urgently needed. We have to think in terms of biological networks: understanding of complex functions may in fact be impossible without taking into consideration influences (rules and constraints) outside of the genome. Systems Biology involves connecting experimental unsupervised multivariate data to mathematical and computational approach than can simulate biologic systems for hypothesis testing or that can account for what it is not known from high-throughput data sets. Metabolomics could establish the requested link between genotype and phenotype, providing informations that ensure an integrated understanding of pathogenic mechanisms and metabolic phenotypes and provide a screening tool for new targeted drug.
A rapid resolution liquid chromatography-tandem mass spectrometry analysis was applied to human u... more A rapid resolution liquid chromatography-tandem mass spectrometry analysis was applied to human urine samples to discover new molecular biomarkers of the alteration of psychophysical well-being due to the environmental, physiological and confinement stress conditions achieved in the MARS500 project. Urine samples of crew members were submitted to metabolomic studies by means of separative techniques coupled with mass spectrometric techniques and data analysis. More than 2,000 metabolite features were found in each comparison, and more than 150 metabolites were identified for every differential analysis. Significant modifications in eight metabolites were shared by the subjects under study.
Vestibular schwannoma (VS) often displays an irregular growth pattern due to factors which remain... more Vestibular schwannoma (VS) often displays an irregular growth pattern due to factors which remain unknown. In this study, VS cell lines from tissue removed during surgery were cultured in order to assess the presence of transforming growth factor beta-1 (TGFbeta-1) a multifunctional polypeptide that has different pleiotropic effects on various tissues. The antibody-binding inhibition assay technique was used. The proliferation rate of VS cell lines in vitro was assessed by [3H]thymidine incorporation, with determinations carried out on days 0, 1, 2 and 4 after removal of fetal calf serum (FCS) from the medium. After 48 h of FCS deprivation, cell growth decreased. TGFbeta-1 release also decreased progressively 24 h after removing FCS from the medium, reaching an overall decrease of 50% after 3 days. The cell culturing procedure appeared suitable for VS cells, in that VS cultures were viable for at least 4 months. TGFbeta-1 was initially shown to be released in significant amounts, but also to decrease progressively to values five times lower after 4 days. These data thus support the possibility of a significant association between TGFbeta-1 release and VS cell replication.
A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization ti... more A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87–96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g−1 and 0.15 mg g−1, respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation.
Background.Evidence has accumulated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is involved in t... more Background.Evidence has accumulated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is involved in the regulation of the proliferation of breast tumor cells. For complete tumor suppression high hypercalcemic doses of 1,25-(OH)2D3 are needed. The aim of this study was to assess the effect of combined treatment of 1,25-(OH)2D3 at low doses and melatonin (MEL) on the proliferation of estrogen-responsive rat breast cancer cell
A sensitive and selective high performance liquid chromatographic doxophylline assay with ultravi... more A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.
Uploads
Papers by Mariano Bizzarri