Search PubMed⌕ Search

Biomedical subjects

S Sassa

Publications and source records attributed to S Sassa.

At least 163 records · Page 9Linked to original sources
PubMed1984

Fluorometric measurement of tin-protoporphyrin in biological samples.

Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, can suppress neonatal and other forms of hyperbilirubinemia in laboratory animals, and represents a potential new approach to the treatment of neonatal jaundice in humans. In order to study the disposition of Sn-protoporphyrin in vivo we have developed a sensitive fluorometric method for the quantitation of this metalloporphyrin in biological samples. The method is sensitive to concentrations as low as 0.01 nmol/ml, and is specific for Sn-protoporphyrin even in the presence of other porphyrins such as protoporphyrin.

Animals↗
PubMed1984

An immunological study of delta-aminolevulinic acid dehydratase specificity consistent with the phylogeny of species.

Rabbit antibody directed to homogeneously purified mouse liver delta-aminolevulinic acid dehydratase cross-reacted with the enzyme in erythrocytes, spleen, kidney and brain in the mouse. The antibody also cross-reacted with the enzyme in the rat, hamster and gerbil, but not in the rabbit, guinea pig, cattle, chick embryo, and human. In contrast, rabbit antibody against the human enzyme partially recognized the monkey enzyme, but not the enzyme in the other species. The species specificity of delta-aminolevulinic acid dehydratase in this study was consistent with the phylogenetic evolution of the species examined.

Animals↗
PubMed1984

Porphyrin-heme biosynthesis in organotypic cultures of mouse dorsal root ganglia. Effects of heme and lead on porphyrin synthesis and peripheral myelin.

Well-myelinated cultures of mouse dorsal root ganglia incubated for 48 h with sigma-aminolevulinic acid (ALA) showed intense porphyrin fluorescence localized in myelin sheaths but not in axons or neuronal somata. When the cultures were continuously incubated with a high concentration of lead, focal swelling and segmental degeneration of myelin began to develop within 2 wk. Incubation of cultures with ALA after 3 wk of lead treatment revealed markedly decreased porphyrin fluorescence in myelin sheaths compared with untreated controls. After 6 wk of lead treatment, myelin showed severe segmental degeneration. Porphyrin fluorescence from ALA at this time was barely detectable in these cultures. No fluorescence was visible in the demyelinated axons; however, silver-impregnation staining after fixation demonstrated continuity of the axon despite the severe loss of myelin. When cultures were continuously incubated with lead and heme together for 6 wk, the segmental demyelination seen in cultures treated with lead alone did not occur. These findings suggest that the lead-induced segmental demyelination in cultured mouse dorsal root ganglia may be due to toxic effects of the metal on the heme biosynthetic pathway in myelinating cells and that exogenous heme may counteract this toxic effect of lead.

Aminolevulinic Acid↗
PubMed1984

Biochemical studies of a patient with hereditary hepatorenal tyrosinemia: evidence of glutathione deficiency.

Metabolic and enzymatic studies in a patient with hereditary tyrosinemia demonstrated for the first time a deficiency of erythrocyte and hepatic glutathione. Markedly decreased hepatic fumarylacetoacetate hydrolase activity was demonstrated in this patient. The activities of hepatic enzymes not involved in tyrosine metabolism were also determined. Assay of mixed function oxidase activity demonstrated low levels of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase, suggesting decreased hepatic detoxification capacity. 5-Aminolevulinic acid dehydratase activity was undetectable. Succinylacetone (4,6-dioxoheptanoic acid), an abnormal metabolic product secondary to fumarylacetoacetate hydrolase deficiency was found in serum and urine. Succinylacetone was demonstrated to inhibit 5-aminolevulinic acid dehydratase in vitro, as did the urine, plasma, and red cell lysates of the patient.

Erythrocytes↗
PubMed1984

Induction of delta-aminolevulinic acid dehydratase in mouse Friend virus-transformed erythroleukemia cells during erythroid differentiation.

The activity of delta-aminolevulinic acid (ALA) dehydratase, an enzyme involved in heme biosynthesis, has been shown to increase in Friend virus-transformed murine erythroleukemia (MEL) cells during erythroid differentiation. In this study, the nature of the increase in ALA dehydratase activity in MEL cells was examined using a monospecific antibody directed to the enzyme. A sevenfold increase in ALA dehydratase activity was observed after cells had been treated with 1.5% Me2SO for 5 days. Ouchterlony double immunodiffusion analysis showed that lysates from untreated and Me2SO-treated MEL cells formed a single precipitin line with rabbit IgG directed to the normal mouse liver ALA dehydratase. A single arc of identity was also observed with the lysates from normal mouse erythrocytes, spleen, liver, and lysates from both uninduced and induced MEL cells. Rocket immunoelectrophoresis demonstrated that lysates from both uninduced and induced cells formed rockets with the IgG and that the peak height of the rocket was proportional to the ALA dehydratase activity applied. The slope of linear plots of rocket peak heights v ALA dehydratase activity was identical for lysates from uninduced and Me2SO-induced cells. Succinylacetone, a potent inhibitor of ALA dehydratase, was shown to markedly inhibit the activity of the enzyme, but did not interfere with the synthesis of ALA dehydratase induced by Me2SO treatment. Me2SO-induced increases in ALA dehydratase activity and the enzyme protein were both blocked by the simultaneous treatment of cells with 5-bromo-2'-deoxyuridine (BrdU). BrdU-mediated repression of ALA dehydratase was partially overcome by treating the cells with thymidine. These data demonstrate that increased ALA dehydratase activity in MEL cells undergoing erythroid differentiation after Me2SO treatment is due to de novo synthesis of the same enzyme protein present in uninduced MEL cells as well as in normal erythrocytes. This represents the first direct demonstration of an increase in a heme biosynthetic pathway enzyme protein in erythroid cells undergoing differentiation.

Animals↗
PubMed1983

ADP-ribosyltransferase activity during the friend virus-induced murine erythroleukemia cell differentiation.

A variety of chemical agents that are known to induce erythrodifferentiation in the Friend virus-induced murine erythroleukemia (MEL) cell have been suggested to mediate DNA cleavage in cultured cells prior to differentiation. The activation of the nuclear enzyme, ADP-ribosyltransferase, depends upon the presence of single strand breaks in DNA. If dimethyl sulfoxide (Me2SO) causes DNA breakage, it would be expected that the activity of ADP-ribosyltransferase would increase. A study of ADP-ribosyltransferase activity during cell growth indicates that both Me2SO-treated and untreated MEL cells exhibit a similar increase in the enzyme activity but the increase in Me2SO-treated cells is delayed by a few hours. When examined at comparable stages of growth, both treated and untreated cells show almost identical levels of enzyme activity. The present data thus do not support the contention that Me2SO induces DNA breakage in the MEL cells.

Animals↗
PubMed1983

Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital and iron on hepatic uroporphyrinogen decarboxylase. Implications for the pathogenesis of porphyria.

Treatment of cultured chick embryo hepatocytes with phenobarbital, polychlorinated biphenyl compounds and 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased delta-aminolaevulinate synthase and decreased uroporphyrinogen decarboxylase activities and porphyrin accumulation; uroporphyrin and heptacarboxyporphyrin predominated. Iron had no effect on these changes. Simultaneous treatment of cultures with dioxin and phenobarbital produced a synergistic response in delta-aminolaevulinate synthase induction, uroporphyrinogen decarboxylase inhibition and porphyrin accumulation. These data suggest that an inhibitor of uroporphyrinogen decarboxylase may be generated in the liver from polychlorinated biphenyl compounds or dioxin by metabolic activation. Additionally these findings bear on the postulated role of these and related chemicals in determining the low levels of uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients.

5-Aminolevulinate Synthetase↗
PubMed1983

[Preparation of progesterone secreting cells from rat ovaries by a density gradient].

Isolation of progesterone secreting cells from the ovaries of immature rats pretreated with pregnant mare serum gonadotropin (PMS) was conducted by a simple procedure which combined the collagenase digestion with a density gradient method. After digestion of the ovarian tissue slices by the enzyme, the residue was gently pressed and placed on a sucrose density gradient. Three bands appearing in the tube after centrifugation were designated as S-1, S-2 and S-3 from the top to the bottom, respectively. The S-1 cells from the ovaries at 6 days after PMS secreted the greatest amount progesterone, i.e. approximately 430 ng per 10(5) cells during the 18th incubation. Progesterone secretion from the S-2 cells was less than 48% of that from the S-1 cells. A physiological interrelation between the S-1 and S-2 cells remains unexplained by the present experiment. The luteal cells were yellow, spheroidal and 15 to 40 mus in diameter. Many vesicle-like particles were found on the cell surfaces. Progesterone secretion from the prepared cells was stimulated significantly by hCG in vitro. This result indicates that progesterone secreting cells isolated by the collagenase-sucrose density gradient preserve their native function as luteal cells. The procedure for preparation of luteal cells in the present report may provide a suitable model for in vitro studies on the corpus luteum.

Animals↗
PubMed1983

Growth-promoting effects of iron- and cobalt- protoporphyrins on cultured embryonic cells.

The effects of hemin (Fe-protoporphyrin) and Co-protoporphyrin on cellular growth have been investigated principally in cultured fibroblasts, but also in myoblasts and hepatocytes from chick embryos. In the presence of horse serum in the culture medium, which by itself did not stimulate cell growth appreciably, Co-protoporphyrin stimulated cell attachment while hemin stimulated cell proliferation of fibroblasts. When Co-protoporphyrin and hemin were added together, the most potent stimulation of cell growth, consisting of increases in cell attachment and rapid cell proliferation, was observed. These findings indicate that the two metalloporphyrins have differential and complementary effects on cellular growth in culture, with synthetic Co-protoporphyrin principally affecting cellular attachment and Fe-protoporphyrin stimulating cellular proliferation.

Animals↗
PubMed1983

DNA fragments in Friend erythroleukemia cells induced by dimethyl sulfoxide.

The idea of the correlation between DNA damage caused by dimethyl sulfoxide (Me2SO) and the induction of erythrodifferentiation in murine erythroleukemia (MEL) cells needs reevaluation as a result of the hydroxyapatite chromatographic analysis of DNA from Me2SO-treated and untreated cells as a function of the time in culture. Because Me2SO causes an arrest in G1 phase and consequent delay in DNA synthesis, the valid comparison would be of the DNA samples taken from control and induced cells at the same stage of growth rather than at the same time in culture. At the same stage of growth, there is no evidence of the induction of DNA damage by Me2SO. Presumably, so-called degradation reported in the past stemmed from the fragments of DNA replication.

Animals↗
PubMed1983

Inhibition of the growth and differentiation of erythroid precursor cells by an endotoxin-induced mediator from peritoneal macrophages.

Conditioned medium from cultures of mouse macrophages incubated with endotoxin in a serum-free medium contains an inhibitor of the growth and differentiation of erythroid precursor cells of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no inhibitory effect. The endotoxin-induced macrophage mediator inhibits the growth and differentiation of dimethyl sulfoxide-, hexamethylenebisacetamide-, butyric acid-, and hypoxanthine-induced cells but has no effect on hemin-induced cells. The conditioned medium has its maximal inhibitory effect on committed erythroid precursor cells, a decreased effect on uncommitted stem cells, and no effect on fully differentiated erythroid cells. These results demonstrate that endotoxin stimulation of macrophages leads to the production of a humoral factor(s) which is critical for the growth and differentiation of erythroid precursor cells.

Animals↗
PubMed1983

Hereditary tyrosinemia and the heme biosynthetic pathway. Profound inhibition of delta-aminolevulinic acid dehydratase activity by succinylacetone.

Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of delta-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 x 10(-7) M to 3 x 10(-7) M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic consequences that mimic another genetic disease, AIP.

Amino Acid Metabolism, Inborn Errors↗
PubMed1983

Metabolism of estradiol in liver cell culture. Differential responses of C-2 and C-16 oxidations to drugs and other chemicals that induce selective species of cytochrome P-450.

The oxidative metabolism of estradiol (the natural estrogen 2,3,5(10)-estratriene-3,17 beta-diol) at positions C-2 and C-16 was examined in primary cultures of chick embryo liver cells using estradiol which was labeled with 3H specifically at either the C-2 or C-16 position as the substrate. Oxidation of estradiol by the cultured liver cells was assessed by the release of 3H which accumulated as 3H2O in the culture medium; both C-2 and C-16 oxidative reactions were detectable in the liver cell cultures by this technique. When incubated with a concentration of estradiol substrate close to the Michaelis constant (Km), approximately 45.8 pmol [2-3H]estradiol and 5.0 pmol [16-3H]estradiol/mg protein per minute underwent oxidative metabolism in untreated cells. Total amounts of oxidized product formation after 2 h of incubation were 28 and 5 pmol/mg protein for C-2 and C-16 oxidation, respectively. Treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide significantly increased oxidation at C-16 (1.9-fold and 2.6-fold greater than control values, respectively), whereas no significant change in C-16 oxidation was observed after treatment of the cultures with 3-methylcholanthrene, benzo[a]pyrene, or benz[a]anthracene. The latter chemicals, however, were found to increase the extent of oxidation at C-2 significantly (i.e., 1.5-2.2-fold increases over control values). The increase in C-2 oxidation after treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide was significantly less than that observed for oxidation at C-16. The apparent Km values for these oxidations in control cultures were 23.5 and 30.3 microM for C-2 and C-16 oxidation, respectively; corresponding maximum velocity (Vmax) values were 119 and 11.7 pmol/mg protein per minute, respectively. These data indicate that the C-2 and C-16 oxidations of estradiol take place in cultured avian hepatocytes and that the extent of metabolism at these positions on the hormone molecule can be altered by chemicals, such as drugs and polycyclic aromatic hydrocarbons, which induce distinctive species of cytochrome P-450 in the liver.

Allylisopropylacetamide↗
PubMed1983

Purification and properties of human erythrocyte uroporphyrinogen decarboxylase: immunological demonstration of the enzyme defect in porphyria cutanea tarda.

The first complete purification of UROD from human erythrocytes and the characterization of the purified enzyme were described. A single enzyme protein catalyzes the four successive sequential decarboxylations of uroporphyrinogen to yield coproporphyrinogen. The enzyme activity is not directly inhibited by iron; however, it is subject to inhibition in liver cells by a number of chemicals, including environmental pollutants such as dioxin. Studies of erythrocyte UROD in PCT patients indicate that the group of patients now defined as having sporadic PCT may represent two different populations; i.e., those who have normal UROD, and those who have decreased UROD in erythrocytes. Genetic heterogeneity of the UROD defect in PCT is also indicated by the identification of both CRM(-) and CRM(+) mutations in this disorder.

Amino Acids↗
PubMed1983

Tin-protoporphyrin suppression of hyperbilirubinemia in mutant mice with severe hemolytic anemia.

Tin-protoporphyrin is a potent competitive inhibitor of heme oxygenase both in vivo in animals and in vitro in isolated enzyme preparations, and when administered to neonatal rats, prevents the development of postnatal hyperbilirubinemia. In this study we examined the effect of the metalloporphyrin on the activity of heme oxygenase in liver, kidney, and spleen, and on the level of bilirubin in plasma in three types of anemic mutant mice with severe hemolytic diseases. We report that the administration of tin-protoporphyrin to anemic mutants homozygous for severe hemolytic disease results in substantial inhibition of heme oxidation in liver, spleen, and kidney and in significant reduction of plasma bilirubin levels. Tin-protoporphyrin thus has the capacity to significantly inhibit in vivo heme degradation and to concurrently diminish plasma bilirubin levels in severe chronic hemolytic disorders.

Anemia, Hemolytic↗
PubMed1982

Purification and properties of bovine spleen heme oxygenase. Amino acid composition and sites of action of inhibitors of heme oxidation.

Microsomal heme oxygenase has been purified from bovine spleen to homogeneity using DEAE-cellulose chromatography, initial hydroxyapatite chromatography, gel filtration, and repeat hydroxyapatite chromatography. The purified enzyme showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of congruent to 31,000. It had a Km for heme of 0.93 microM, a specific activity of 6770 units/mg of protein, and a turnover number of 3.5 mol/mol of enzyme/min. Amino acid analysis of the purified enzyme revealed an abundance of glutamine and glutamic acid residues, a log tryptophan content (2 tryptophan residues/mol of enzyme), and four free sulfhydryl groups, only two of which were accessible to sulfhydryl (--SH) inactivating reagents without unfolding of the protein. Consistent with these findings, the purified enzyme had a relatively low extinction coefficient at 280 nm (E11%cm = 8.12) and was relatively resistant to inactivation by --SH inactivating reagents. NADPH-cytochrome c reductase from bovine liver was purified to homogeneity and biliverdin reductase from bovine spleen was partially purified. The heme oxygenase system was reconstituted from preparations of all three purified enzymes and, utilizing this reconstituted system, the specific sites of the inhibitory actions of --SH inactivating reagents, inorganic metals, and metalloporphyrins on the heme degrading sequence of reactions were examined. Sn-, Co-, Zn-, and Mn-protoporphyrin strongly inhibited heme degradation in a competitive manner. The Ki values for Sn-, Co-, and Zn-protoporphyrin were determined to be 0;033, 0.082, and 0.13 microM, respectively. Mg-, Ni-, and Cu-protoporphyrin had little effect on heme degradation by the reconstituted system. Metals such as Pt2+ and Hg2+ strongly inhibited the activity of the reconstituted heme oxygenase system, but the principal site of action of these metals was at the level of NADPH-cytochrome c reductase or biliverdin reductase. Similarly --SH inactivating reagents, such as p-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide inhibited the reaction catalyzed by the reconstituted heme oxygenase system principally by inhibiting the activity of NADPH-cytochrome c reductase.

Amino Acids↗