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A Iannone

Publications and source records attributed to A Iannone.

At least 37 records · Page 2Linked to original sources
PubMed1990

Selective uptake of 2-thiouracil into melanin-producing systems depends on chemical binding to enzymically generated dopaquinone.

2-Thiouracil (TU), an antithyroid drug, is receiving growing interest as a specific tumor marker for malignant melanoma, owing to its capability of being selectively accumulated into active melanin-producing tissues. However, up until now, the molecular mechanism of TU uptake by growing melanin has remained largely unknown. In an attempt to fill this gap, we have investigated the effect of TU on the tyrosinase catalyzed oxidation of tyrosine. At a concentration of 0.5 mM, TU was found to totally inhibit melanin formation by tyrosinase catalyzed oxidation of 0.25 mM tyrosine in phosphate buffer at pH 6.8. Polarographical monitoring of oxygen consumption under conditions of complete suppression of melanogenesis revealed a significant tyrosinase activity, with TU acting as a modest non-competitive inhibitor of the enzyme (Ki = 0.6 mM). HPLC and TLC analysis of the tyrosine-tyrosinase reaction in the presence of excess TU showed that the substrate is progressively consumed and a major hitherto unknown product (lambda max = 284 nm), positive to ninhydrin and ferric chloride, is concomitantly formed. This was isolated by repeated gel filtration chromatography of the reaction mixture on Sephadex G-10 and was formulated as the TU-dopa adduct 3,4-dihydroxy-6-(4'-hydroxypyrimidinyl-2'-thio)phenylalanine by spectral analysis. These results suggest that selective TU incorporation in pigmented melanomas and other melanin-producing systems is due to the covalent binding to dopaquinone, produced by tyrosinase catalyzed oxidation of tyrosine.

Animals↗
PubMed1990

Metabolism of nitroxide spin labels in subcellular fraction of rat liver. I. Reduction by microsomes.

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.

Animals↗
PubMed1990

Metabolism of nitroxide spin labels in subcellular fractions of rat liver. II. Reduction in the cytosol.

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of five nitroxides in cytosol derived from rat hepatocytes. The nitroxides were chosen to provide information on the effects of the type of charge and the ring on which the nitroxyl group is located. The rates of reduction were fastest for a six-membered positively charged nitroxide ('CAT-1') and slowest for an anionic five-membered ring nitroxide ('PCA'). Changing levels of glutathione, sulphydryl groups in general, NADPH or NADH had little or no effect on the rates of reduction, while the addition of ascorbate oxidase essentially abolished reduction of the nitroxides. The products of reduction by the cytosol were the corresponding hydroxylamines. The overall rates of reduction of neutral or anionic nitroxides were much slower than those observed with intact cells. We conclude that the primary source of metabolism of nitroxides by cytosol is reduction by ascorbate and that under most conditions reduction of nitroxides in the cytosol is not a major factor in the metabolism of nitroxides by cells.

Animals↗
PubMed1989

Metabolism in rat liver microsomes of the nitroxide spin probe tempol.

Paramagnetic nitroxide spin labels have been extensively used to probe various biophysical and biochemical properties of the cellular environment. Recently nitroxides have been proposed as contrast enhancing agents in proton magnetic resonance imaging and contrast enhancement has been demonstrated in animal studies. Nitroxides, possessing a stable unpaired electron, increases the relaxation rates of protons, providing an enhancement of contrast. Nitroxides are metabolized intracellularly principally via reversible reduction to hydroxylamines. Rates of reduction depend on the physical characteristics of the nitroxides, in general 5-membered pyrrolidine ring are reduced more slowly than those with a 6-membered piperidine ring. Oxidation back to the nitroxide is relevant for lipid soluble hydroxylamines, while is low for water soluble ones. It is known that nitroxides are metabolized by subcellular fractions (cytosol, mitochondria, microsomes), though the enzymatic and non-enzymatic systems involved are poorly characterized. In the present study, the first of the necessary steps toward a systematic study of the metabolism of nitroxides by subcellular organelles, we have chosen to study the metabolism of 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl in isolated rat liver microsomes. Microsomes were able to reduce Tempol slowly without any substrate addition; when NADPH was added, the reduction rate substantially increased. In phenobarbitone induced rats the reduction rate was significantly higher than in not-induced microsomes. NADPH-dependent reduction rate was inhibited by thallium chloride (an inhibitor of the flavin-centered cytochrome P-450 reductase), superoxide dismutase, and by N-ethylmaleimide; menadione increased it. The Tempol reduction rate was not significantly affected by various cytochrome P-450 inhibitors with the sole exception of metyrapone. A solution containing purified cytochrome P-450 reductase and NADPH readily reduced Tempol. Microsomes fortified with NADPH were able to reduce Tempol at an appreciable rate. In order to distinguish between reduction of nitroxides to hydroxylamine or destruction of nitroxides following nitroxide reduction, microsomal suspensions were treated with a mild oxidant (ferricyanide 0.5-10 mM). The recovery varied from 40 to 60%, indicating a process of probe destruction leading to as yet unknown metabolites. The present study clearly indicates that, in this model system, cytochrome c (P-450) reductase and not cytochrome P-450 is responsible for the observed Tempol metabolism; along with hydroxylamine formation, other Tempol derived metabolites are formed during the process.

Animals↗
PubMed1989

Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides.

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides.

Air↗
PubMed1989

Bone marrow uptake of liposome-entrapped spin label after liver blockade with empty liposomes.

Using an ESR spectrometer, we studied the time course of the uptake of the liposome-entrapped spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylammonium in liver, spleen, and bone marrow following reticuloendothelial liver blockade. Our results show that suppression of the phagocytic activity of the liver increases the delivery of liposomes to the spleen and bone marrow without substantially altering uptake by the liver.

Animals↗
PubMed1989

A modified roller tube technique for organotypic cocultures of embryonic rat spinal cord, sensory ganglia and skeletal muscle.

The roller tube technique as initially described in the literature in 1981, was modified in several aspects for the coexplantation of embryonic rat spinal cord with attached dorsal root ganglia and skeletal muscle from newborn rats. The high metabolic activity of this coculture system required a particular culturing protocol to stabilize pH and osmotic pressure. The appropriate adjustment of the partial pressure of carbon dioxide gas in the incubator proved to be essential for the control of the pH within narrow limits (7.3 +/- 0.1). The adjustment of the osmotic pressure of the medium (290-300 mOsm) improved the growth of the cultures considerably. Roller drum speed was set to 120 revolutions per hour for enhanced flattening of the culture. A simple rating system was used to evaluate neuronal and non-neuronal outgrowth under different modifications of the culture system. Furthermore, morphological and electrophysiological criteria were defined for evaluating individual neurons. The technique described insures the growth of long-term organotypic cocultures of spinal cord, sensory ganglia and skeletal muscle.

Animals↗
PubMed1989

Free radical activation of monomethyl and dimethyl hydrazines in isolated hepatocytes and liver microsomes.

Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethyl-hydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with CoCl2 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines.

Animals↗
PubMed1989

Free radicals and lipid peroxidation in liver of rats kept on a diet devoid of choline.

Rodents kept on a choline devoid (CD) diet up to 14 months develop hepatic lesions progressing through two broad stages. The first is characterized by severe steatosis and increase in cell turnover, the second by a gradual clearance of the deposited fat and fibrosis. Hepatocellular carcinomas eventually arise in rats fed for over 12 months, even though the animals aer not exposed to chemical carcinogens. It has been suggested that the diet may trigger generated thereby may be responsible for initiation of liver cancer and promotion. The radicals would lead to DNA damage, and the altered DNA in a proliferating liver would result in initiation of the carcinogenic process. In this communication we present evidence that the diet used in the above studies contained stable fatty acid isomers with conjugated dienes, which are absorbed and deposited in rat liver. This finding cast doubts on whether a CD diet does indeed cause a peroxidation of cellular membrane lipids. Electron spin resonance (ESR) spectroscopy was also used to investigate whether any abnormal pattern of free radicals exists in the liver of rats fed a CD diet. No significant differences were noted in ESR spectra of either transition metal-centered signals, or organic free radicals.

Animals↗
PubMed1987

Free-radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation.

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.

Animals↗
PubMed1983

Phenotypic immaturity of T and B lymphocytes in cord blood of full-term normal neonates. Analysis of cell surface markers by using conventional techniques and monoclonal antibodies.

We examined mononuclear cell subsets in cord blood of normal newborns by surface marker analysis. The percentages of T lymphocytes (E-rosetting and T3+ cells) were lower in cord blood than in peripheral blood (PB) from adults, while the percentage and absolute number of T6+ cells were higher in cord blood. As the sum of T4+ and T8+ cells exceeded the values of E-rosetting lymphocytes in cord blood, we suggest that immature lymphocytes with the phenotype of 'common' thymocytes (T6+, T4+, T8+) are present in cord blood of full-term newborns. Higher percentage and absolute number of B lymphocytes were detected in cord blood. More than 50% of B cells in cord blood formed rosettes with mouse erythrocytes, a surface marker of functional immaturity. Finally, cells bearing receptors for IgG-Fc fragments or C3 and expressing Ia-like and M1 antigens were uniformly increased in cord blood, suggesting higher percentages of cells of the monocytic lineage.

Adult↗
PubMed1983

Analysis of mononuclear cell subsets in pregnancies with intrauterine growth retardation. Evidence of chronic B-lymphocyte activation.

We have examined the mononuclear cell (MC) subpopulations of 5 pregnant women: Three of them had a previous history of IUGR, whereas two were primiparae and presented IUGR at the term of gestation. IUGR was confirmed after delivery in three women. The analysis of MC subsets was performed by rosetting and immunofluorescence techniques; both heterologous antisera specific for human immunoglobulins and monoclonal antibodies specific for T cell antigens and for M1 and Ia-like antigens were used. The three patients with IUGR confirmed at birth presented numbers of circulating lymphoid cells positive for cytoplasmic IgM, IgG and IgA and with morphologic features of plasmablasts or plasma cells at least tenfold higher than in normal pregnant women at the term of gestation. Our data suggest that chronic activation of the lymphoid system occurs in pregnant women with IUGR. Maternal abnormal reactivity to fetal antigens or to undiagnosed chronic infections may be likely explanations for this phenomenon. Further studies are clearly needed to clarify the relationship between B-lymphocyte activation and pregnancy and to examine the hypothesis of an altered balance of the immunoregulatory T lymphocyte subsets in patients with IUGR.

B-Lymphocytes↗
PubMed1980

Antagonist EMG temporal patterns during rapid voluntary movement.

This study of 10 normal college-aged women was designed to clarify possible antagonist control mechanisms during the silent period of the agonist in rapid elbow extension tasks. Antagonist electromyographic temporal patterns were observed after agonist silence under various conditions to determine if antagonist activity in the rapid movement was controlled supraspinally (preprogrammed), spinally (reflexively), or by a combination of the two mechanisms. The subjects followed a velocity-controlled dot displayed on an oscilloscope. The antagonist latencies remained constant during intentionally and unintentionally terminated movements, but were altered by load conditions. This was seen as an automatic deceleration response, elucidating differences between antagonist control during ballistic and rapid movements.

Action Potentials↗
PubMedDate not supplied

NMR study of water exchange across the hepatocyte membrane.

An understanding of the cellular permeability for water is needed to evaluate MR images of complex tissues, such as liver, and to interpret the effects of contrast agents. To obtain data essential for such an understanding we measured water exchange across the isolated rodent hepatocyte membrane by proton NMR relaxation with dextranmagnetite as a relaxation agent. The results are treated as water exchange in a two-compartment system, and possible reasons for deviations from that behavior are analyzed. The mean residence time of intracellular water was approximately 40 ms at 37 degrees C. We found the lower limit for the diffusional permeability of the hepatocyte membrane to be 8 x 10(-3) cm s-1. These results, combined with consideration of hepatic anatomy indicate that the failure to observe effects on the T1 of liver from particulate contrast agents such as magnetite, Gd-starch, and liposome encapsulated Mn2+ is due to the localization of these agents in the Kupffer cells. Also, the nonexponential T1 decay observed in normal liver is unlikely to be due to slow exchange of water between compartments.

Animals↗