Biomedical subjects
Publications and source records attributed to M A Grillo.
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.
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.
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.
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.
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.
The sural nerve is generally used as a graft in nervous reconstructions due to its easy access and minimal sequelae after its withdrawal. Although this nerve presents a constancy in its topographical localization, anatomical variations are frequent. Thus, trying to determine the formation pattern of the sural nerve complex, 38 legs from 19 cadaveres were dissected. The data obtained differ from those of the literature as to the frequency of the occurrence of the two classically known patter (pattern I and II). Besides, it was possible to identify two other paterns (pattern III and IV) of the sural nerve formation not yet described The cutaneous nerve and the comunicating peroneal nerve were found in 21% of the cases. In the literature, the frequency is of 65.8% to 80%. Pattern II, where the sural nerve is originated from the medial sural cutaneous nerve, was identified in 52.6% of cases, while in the literature its frequency is of 20 to 34.2%. Pattern III, not yet described in the literature, had the sural nerve coming from the junction between the medial sural cutaneous nerve and the lateral sural cutaneous one, with a frequency of 21% of the cases. Pattern IV, also not yet described in the literature, had the sural nerve originated from the common fibular nerve in 5.2% of patients. It was also possible to identify in the sample the presence of the lateral sural cutaneous nerve (branch of the common fibular nerve) and of the peroneal communicating nerve (branch of the lateral sural cutaneous nerve) with anatomical characteristics indicating them as an alternative source of nerve grafts.
In hepatocytes in culture, ornithine aminotransferase activity remained higher when the cells were cultured at low oxygen tension (5%) than at high tension (21%), that is, it was higher in hepatovenous conditions. Northern blot analysis showed that the amount of the specific mRNA for the enzyme was also higher. Results of experiments performed in the presence of CoCl2, to replace the central Fe2+ in heme, or succinylacetone, to inhibit heme synthesis, support the view that a heme protein participates in the regulation of ornithine aminotransferase activity by oxygen. The oxygen sensor does not appear to act through phosphorylation by kinase C, as TPA has no significant effect on the process, but a phosphorylation dependent on cAMP might be involved.
The nicotine metabolite cotinine was administered to abstinent cigarette smokers to determine whether it has pharmacologic activity as assessed by various physiologic and subjective measurements. By means of a randomized, double-blind, placebo-controlled counterbalanced-order design, subjects received cotinine base (30 mg) intravenously after 48 hours of abstinence from cigarette smoking. Serum cotinine concentrations increased to levels commonly achieved during daily cigarette smoking, whereas no change in serum nicotine concentration was observed. Cotinine compared with placebo produced subjective differences in self-reported ratings of restlessness, anxiety and tension, insomnia, sedation, and pleasantness. Cotinine had minimal effects on cardiovascular measurements. These findings indicate that cotinine is behaviorally active in the setting of cigarette abstinence at blood concentrations similar to those commonly achieved through daily cigarette smoking.
1. Berenil, administered to rats in vivo, promoted a decrease in liver SAMDC activity, but an increase in ODC and SAT activity. 2. Its effect on ODC was completely prevented by cycloheximide, that on SAT only partially. 3. Berenil had no effect on ODC activity in adrenalectomized rats. Adrenergic antagonists counteracted the effect of Berenil on ODC activity. 4. Polyamine content was increased. The maximum modification was observed for putrescine and N1-acetylspermidine.
1. Putrescine and spermidine content increased in hepatocytes during culture. In the presence of 10 microM Berenil, putrescine content was further increased, while the increase of spermidine was prevented. 2. Ornithine decarboxylase activity was markedly reduced, and to a lesser extent also S-adenosylmethionine decarboxylase activity. 3. Berenil appears to promote an increase in the transformation of spermidine into putrescine, and to inhibit the polyamine efflux.