Biomedical subjects
Publications and source records attributed to S Colombatto.
S-adenosylmethionine is the major methyl donor in all living organisms, but it is also involved in many other reactions occurring through radical-based catalysis. The structure and function of some of these enzymes, including those involved in the synthesis of the molybdenum cofactors, biotin, lipoate, will be discussed.
BACKGROUND AND AIMS: Myofibroblast-like cells, originating from activation of hepatic stellate cells (HSC/MFs), play a key role in liver fibrosis, a potentially reversible process that may rely on induction of HSC/MFs apoptosis. While this possibility has been shown in cultured rat HSC, very limited data are currently available for human HSC/MFs. METHODS: Cultured human HSC/MFs were exposed to several proapoptotic stimuli, including those known to induce apoptosis in rat HSC/MFs, and induction of cell death and related mechanisms were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: In this study we report that fully activated human HSC/MFs did not undergo spontaneous apoptosis and survived to prolonged serum deprivation, Fas activation, or exposure to nerve growth factor, tumour necrosis factor alpha (TNF-alpha), oxidative stress mediators, doxorubicin, and etoposide. Induction of caspase dependent, mitochondria driven apoptosis in HSC/MFs was observed only when protein synthesis or transcription were inhibited. Importantly, the process of HSC activation was accompanied by changes in expression of a set of genes involved in apoptosis control. In particular, activated human HSC/MFs in culture overexpressed Bcl-2. The role of Bcl-2 was crucial as Bcl-2 silenced cells became susceptible to TNF-alpha induced apoptosis. Finally, Bcl-2 was markedly expressed in HSC/MFs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis. CONCLUSIONS: Human activated HSC/MFs are resistant to most proapoptotic stimuli due to Bcl-2 overexpression and this feature may play a key role in the progression of fibrosis in chronic liver diseases.
BACKGROUND AND AIMS: Activated myofibroblast-like cells, originating from hepatic stellate cells (HSC/MFs) or other cellular sources, play a key profibrogenic role in chronic liver diseases (CLDs) that, as suggested by studies in animal models or rat HSC/MFs, may be modulated by reactive oxygen intermediates (ROI). In this study, human HSC/MFs, exposed to different levels of superoxide anion (O(2)(.-)) and, for comparison, hydrogen peroxide (H(2)O(2)), were analysed in terms of cytotoxicity, proliferative response, and migration. METHODS: Cultured human HSC/MFs were exposed to controlled O(2)(.-) generation by hypoxanthine/xanthine oxidase systems or to a range of H(2)O(2) concentrations. Induction of cell death, proliferation, and migration were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: Human HSC/MFs were shown to be extremely resistant to induction of cell death by O(2)(.-) and only high rates of O(2)(.-) generation induced either necrotic or apoptotic cell death. Non-cytotoxic low levels of O(2)(.-), able to upregulate procollagen type I expression (but not tissue inhibitor of metalloproteinase 1 and 2), stimulated migration of human HSC/MFs in a Ras/extracellular regulated kinase (ERK) dependent, antioxidant sensitive way, without affecting basal or platelet derived growth factor (PDGF) stimulated cell proliferation. Non-cytotoxic levels of H(2)O(2) did not affect Ras/ERK or proliferative response. A high rate of O(2)(.-) generation or elevated levels of H(2)O(2 )induced cytoskeletal alterations, block in motility, and inhibition of PDGF dependent DNA synthesis. CONCLUSIONS: Low non-cytotoxic levels of extracellularly generated O(2)(.-) may stimulate selected profibrogenic responses in human HSC/MFs without affecting proliferation.
The enzymes responsible for protein methylation by S-adenosylmethionine, both at the carboxyl groups and at the nitrogen groups, are reviewed. The possibility that the reactions involved may be reversible is also considered.
Arginine is a precursor of proteins and employed in urea synthesis. It is also the precursor of many other compounds, such as creatine, nitric oxide, polyamines, agmatine, proline. In this review, its transport and that of other basic amino acids are examined, along with its transformation into nitric oxide, agmatine and proline, and the mutual regulation of the individual pathways.
Recently agmatine, decarboxylated arginine, has been shown to be an important biological compound in several animal tissues. This paper summarizes the known information regarding the transport of arginine, its decarboxylation and the effects of the agmatine formed mainly on NO and polyamine synthesis.
The DNA-binding properties of cystamine compared with natural occurring polyamines have been studied in vitro by means of ethidium bromide displacement assays, studies of DNA thermal stability and analyses of DNA-B/DNA-A transition. While the first two methods did not put in evidence any peculiar property in the binding capability of cystamine, CD studies showed the interesting ability of cystamine to shift the equilibrium B/A-DNA towards the B-form. In the same experimental conditions spermine and spermidine induced the A form of DNA, instead putrescine and cadaverine did not show any particular activity. The ability of cystamine to bind DNA, as shown also by its DNA radioprotective capability, might be important in chromatin condensation and stabilization, and might be a cause of the antiviral activity observed by some authors.
BACKGROUND/AIMS: Agmatine, the compound formed by decarboxylation of arginine, is believed to be an endogenous neurotransmitter through interaction with the imidazoline receptors. However, it also appears to regulate rat hepatocyte polyamines by modifying both their synthesis and their catabolism. As the decrease in polyamine content has been correlated with apoptosis, we examined the possibility that agmatine has an effect on this phenomenon. METHODS: Apoptotic cells were detected by visualizing nuclear shrinkage/fragmentation in hepatocytes cultured at 21 and 5% oxygen tension. Caspase-3 activity, cleavage of PARP, release of cytochrome c and mitochondrial swelling were therefore measured in the two conditions and in the presence or not of agmatine. RESULTS: In rat hepatocytes agmatine promoted apoptosis, procaspase 3 processing and increase of caspase-3 like activity. This occurred through mitochondria swelling and release of cytochrome c. Cyclosporin A and catalase blocked the swelling. CONCLUSIONS: Our experiments show that agmatine, besides all the known biological effects, has also part, at least in hepatocytes, in the modulation of programmed cell death.
In the central nervous system, NOS activity is involved in several physiological events, such as refinement of afferent connections in development, or linking cerebral blood flow to neural activity in adulthood, and also in many pathological events, such as cell death in brain ischemia and regulation of vasospasm in hemorrhage. Therefore, we studied NOS activity in the CNS. We describe a fast and accurate method in which we use HPLC analysis to identify and quantify citrulline eluted by ion-exchange chromatography, thus implementing the current method to evaluate NOS activity. This technique could be readily applied for NOS activity determination not only in brain, but also in all other tissues.
Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mM; Vmax = 30 pmol x min x (mg protein)-1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.
A series of diamines with the general structure NH2(CH2)xNH2, x=2-12, was tested for their potential effects on cell proliferation of cultured rat C6 glioma cells in comparison to natural polyamines. Long chain diamines reduced cell number after 48 h in culture with a sequence of 1,12-diaminododecane (1,12-DD) >1,10-diaminodecane >1,9-diaminononane. Polyamines (putrescine, spermidine and spermine) as well as diamines up to a CH2-chain length of x=8 were found to be ineffective. The spermine analogue 1,12-DD was the most effective molecule in reducing cell number in an irreversible, dose-dependent manner (EC50=3 microM under serum-free conditions). In further experiments we investigated the mechanisms of action of 1,12-DD. The compound had only a minor effect on cell cycle and did not affect free internal calcium concentration. Under physiological conditions 1,12-DD interacts with triplex DNA but not with duplex DNA. Ornithine decarboxylase activity as well as the concentration of internal polyamines were found to be reduced by 1,12-DD. Polyamine application, however, was not able to reverse the effect of 1,12-DD, indicating a polyamine-independent or non-competitive mechanism of action. 1,12-DD reduced cell number by induction of apoptosis as well as necrosis. In molecular modeling studies it was found that a minimal hydrophobic intersegment of at least 4 A was required to make a diamine an effective drug in respect to cellular growth. A hydrophobic gap of this size fits the minimum requirement expected from molecular modeling to provide space for hydrophobic interactions with parts of proteins like a CH3-group. Our results show that 1,12-DD acts as a potent drug, reducing the number of C6 glioma cells, and suggest that its spatial and hydrophobic properties are responsible for its mechanism of action.
BACKGROUND/AIMS: The human iNOS promoter contains a consensus sequence for binding the hypoxia inducible factor. The aim of this study was to see whether iNOS gene expression is triggered by oxygen tension in rat hepatocytes exposed in vivo to high (periportal) and low (perivenous) oxygen tension. METHODS: Hepatocytes transfected or not with a plasmid containing rat iNOS promoter linked to chloramphenicol acetyltransferase were cultured at 21% and 5% oxygen tension. In normal hepatocytes, iNOS protein, mRNA and activity were detected. In transfected cells, chloramphenicol acetyltransferase activity was measured. RESULTS: In cells cultured in a hypoxic environment, both iNOS protein and mRNA increased, whereas the nitrite level in the medium decreased. However, electron paramagnetic resonance analysis and in vitro iNOS activity indicated that iNOS was active. Transfection experiments showed that the expression of chloramphenicol acetyltransferase driven by iNOS promoter was increased in cells maintained at low oxygen tension. CONCLUSIONS: Our experiments show that in rat hepatocytes: 1) iNOS is induced by low oxygen tension; 2) the modification occurs at the transcriptional level; 3) the enzyme at 5% oxygen is able to catalyze the synthesis of NO, although no nitrites are accumulated in the medium. These findings could have physiopathological relevance, e.g. in determining the resistance of perivenous hepatocytes to ischemia injury.
This work is the first demonstration that cystamine is actively accumulated in spheroplasts of Saccharomyces cerevisiae. We have identified and quantitatively determined the transported cystamine in extracts of spheroplasts that have been incubated over different time periods and in the presence of different amounts of cystamine. The method used, already reported in literature for the identification of natural aliphatic polyamines in biological fluids, consists of a derivatization of spheroplast extracts with dabsyl-chloride and subsequent chromatographic analysis in HPLC. Our results show that cystamine accumulation is a function of time, it increases up to 2.5 min then decreases. Transport is inhibited by natural aliphatic polyamines, which, at the same concentration of cystamine (1 mM), cause a decrease in cystamine transport of about 90% for spermidine, 50% for spermine and only 15% for putrescine. Furthermore, transport is energy-dependent as demonstrated by a significant decrease observed in the presence of 2,4-dinitrophenol, ouabain and vanadate. In particular 0.2 mM ouabain causes a decrease of more than 60% in cystamine transport. Our data suggest that cystamine is transported in Saccharomyces cerevisiae spheroplasts via the same polyamine transport system(s) known to be operating in higher eukaryotic cells.
Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.
Casein kinase 2 purified from human erythrocyte cytosol has been found to phosphorylate human spermidine/spermine N1-acetyltransferase (SSAT) expressed as a fusion protein in E. coli and purified to homogeneity with a specific activity similar to that reported for pure human SSAT. The amino acid sequence of the protein revealed not less than four phosphorylable residues, optimal target for protein kinase 2 phosphorylation being flanked by acid residues in position +1 and +3. Our results indicate that most 32P-phosphate is taken up by Ser residues, as evidenced by HCl hydrolysis and electrophoresis and that the phosphorylation extent is modulated by the physiological polyamine concentration. Partial digestion with trypsin at a low concentration for less than one hour preferentially hydrolyzes Lys-Arg-Arg in position 141-143 of the SSAT suggesting that the Ser-phosphorylated residues are located in the C-terminus of the protein, probably Ser 146 and 149.
Uptake of polyamines has been investigated extensively in many cells, but not in placenta, where the polyamine-polyamine oxidase system is supposed to have an immunoregulatory function in pregnancy. Due to the importance of the transfer in this tissue, we have started this study. JAR human placental choriocarcinoma cells in monolayer at confluency were used as a model for measuring the key enzymes of polyamine synthesis and interconversion, rate of uptake and efflux, and the polyamine content. Polyamines were taken up by JAR cells and released by an independent mechanism. Ornithine decarboxylase and spermidine acetyltransferase activities and the rate of transport in and out of the cell were much higher than in other cells, such as L1210 cells. However the systems used for uptake and release appear in many respects to be similar to those observed in L1210 cells, but different from others. The uptake appears to be regulated by an inhibitory protein. Moreover, protein kinase C appears to be involved in the process. The efflux also is regulated as in L1210 cells, through control of H+ and Ca2+ concentration. In conclusion, this study shows that, in JAR cells, ornithine decarboxylase and spermidine acetyltransferase activities were much higher than in other cells, and so was the rate of transport in and out of the cells. As a result, a much higher polyamine content was observed.
To understand the mechanism involved in the liver zonation of polyamines, we have studied the possible role of oxygen tension. When hepatocytes were cultured at 21% and at 5% oxygen in atmosphere to mimic periportal and perivenous conditions, polyamine content was modified. The observed modifications suggested an effect on the interconversion pathway. Spermidine acetyltransferase (SAT) activity and N1-acetylspermidine were therefore measured in the same conditions. SAT activity was markedly increased after 6 hours and N1-acetylspermidine was accumulated in the cells. This was caused by new enzyme synthesis. The higher expression of SAT was accompanied by an increase in the content of the specific messenger RNA (mRNA). When liver cells were depleted of polyamines, SAT activity and the specific mRNA content were not enhanced by oxygen deprivation, but they increased when polyamines were added again. Polyamines therefore appear to be necessary to promote the increase in SAT mRNA.
Reduced interleukin 2 (IL-2) production has been described as one of the most frequent immune dysfunctions observed at relapse in patients with disseminated solid neoplasms. It was seen that patients treated for breast cancer (NED-no evidence of disease- at check-up) have a relapse percentage of 4.7% if the IL-2 plasmatic level is normal while the relapse percentage increased to 33.3% if it is low after a 10-12 month follow-up. This link between low IL-2 and the host immune response is a new prognostic indicator and one not strictly related to the tumour itself.