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Biomedical subjects

A De Flora

Publications and source records attributed to A De Flora.

At least 109 records · Page 6Linked to original sources
PubMed1982

Selective deactivation of ICR mutagens as related to their distinctive pulmonary carcinogenicity.

The mutagenicity patterns and the metabolic behaviour of 4 structurally related ICR compounds were investigated using the Salmonella/microsome test. ICR 191 and ICR 170 reverted 4 his- strains (TA1537 greater than TA100 greater than TA98 and TA1538), while ICR 191-OH and ICR 170-OH reverted TA1537 only, with a narrow range of mutagenic activity. Although all the products tested were shown to contain a common mutagenic impurity, analysis of chemicals fractionated by h.p.l.c. provided evidence that pure ICR 191-OH and ICR 170-OH also induce frameshift errors. The mutagenic activity of all the 4 ICR compounds, as well as of their common impurity, was decreased in the presence of S-9 mix containing rat liver S-9 fractions, a trend confirmed by h.p.l.c. of ICR/S-9 mixtures. Despite the greater mutagenic potency in the absence of metabolic systems, ICR 191 was deactivated far more efficiently and rapidly than ICR 170 by a variety of mouse (liver greater than lung) and rat (liver greater than testis greater than kidney greater than lung greater than striated muscle greater than spleen) S-9 fractions. Mouse preparations were more effective than the corresponding rat preparations. Aroclor 1254 strongly stimulated tha activity of th NADPH-requiring enzymes responsible for ICR deactivation. H.p.l.c. analyses ruled out interconversion among the 4 ICR compounds in the presence of liver S-9 fractions, while revealing the appearance of new distinct peaks for ICR 191, ICR 170 and ICR 170-OH. The selective metabolic deactivation of ICR compounds may be related to the findings of previous carcinogenicity studies (1-3) which showed the induction of lung adenomas following i.v. but not i.p. administration of ICR 170. Conversely, ICR 191 and ICR 191-OH showed no activity even when administered i.v. Since intercalation of ICR compounds into DNA is the molecular basis for both mutagenic and antitumor activities (4), their distinctive metabolic reactivity may be also responsible for the differential efficacy in pharmacological assays.

Aminacrine↗
PubMed1981

Decay of proteinase and peptidase activities of human and rabbit erythrocytes during cellular aging.

Variations in activity of the membrane-bound and cytosolic proteinases and peptidases were analyzed in human and rabbit erythrocytes at various stages of their life-span. The patterns observed with human erythrocytes were the following. (a) The acidic endopeptidase activity associated with the membranes undergoes a substantial decline during cellular aging, with an estimated half-life of 65 days. Concomitantly it appears to become progressively more latent. (b) All cytosolic proteinase and peptidase activities described previously (Pontremoli, S., Melloni, E., Salamino, F., Sapartore, B., Michetti, M., Benatti, U., Morelli, A. and De Flora, A. (1980) Eur. J. Biochem. 110, 421-430) decline exponentially throughout the erythrocyte life-span, with the exception of dipeptidyl aminopeptidase III. The calculated half-lives were: 60 days for the neutral endopeptidase; 87 days for the total acidic endopeptidase activity which is accounted for by three distinct enzymes; 49 days for aminopeptidase B and 133 days for a second aminopeptidase with broad substrate specificity; 84 days for dipeptidyl aminopeptidase II. The results obtained with the rabbit erythrocytes were: (a) no significant decline of leucine aminopeptidase, dipeptidyl aminopeptidase II and III activities in the transition from reticulocytes to mature erythrocytes; (b) very limited decline of aminopeptidase B activity; (c) a pronounced age-dependent decay, in increasing order, of neutral endopeptidase, aminopeptidase A, carboxypeptidase and acidic endopeptidase activities.

Aminopeptidases↗
PubMed1981

Variation of the frameshift activity of a mutagen (ICR-191) following nitrosation in human gastric juice.

The acridine half-mustard mutagen ICR-191 elicits peculiar frameshift mutations in S. typhimurium, which are greatly decreased in the presence of rat liver preparations. Following in vitro preincubation of ICR-191 with sodium nitrite and human gastric juice, these patterns of mutation are replaced by frameshift errors requiring metabolic activation and showing a different molecular specificity.

Aminacrine↗
PubMed1981

G6PD Napoli and Ferrara II: two new glucose-6-phosphate dehydrogenase variants having similar characteristics but different intracellular lability and specific activity.

Two new glucose-6-phosphate dehydrogenase (G6PD, D-glucose 6-phosphate: NADP oxido reductase, E.C. 1.1.1.49) variants, designated G6PD Napoli and G6PD Ferrara II, are described in propositi from two unrelated families. Characterization side by side of the two variants according to W.H.O. recommendations reveals minor differences which are mostly related to utilization of artificial substrates (increased in both cases as compared with normal G6PD type B). Other properties, which are not significantly distinctive between the two variants, are an enzyme activity amounting to nearly 20% of normal, a decreased electrophoretic mobility, decreased Km values for glucose-6-phosphate and NADP, normal thermostability and biphasic pH curves. However, marked differences emerged between the two variants and between either variant and G6PD B as well, when a number of microtechniques were used. These were: (1) the half-lives of G6PD Napoli and G6PD Ferrara II are 16 and 29 d, respectively, while that of G6PD B is 63 d; (2) the specific activities, measured by a method involving direct estimation of G6PD protein on sodium dodecyl sulphate polyacrylamide gel electrophoretic tracings, are 166 I.U./mg (G6PD Napoli) and 59 I.U./mg (G6PD Ferrara II), as compared with normal value of 180 I.U./mg (G6PD B). On the whole, these findings allow the conclusion that the deficiency of catalytic activity is related to an accelerated though distinctive decay of both mutant enzyme proteins within the affected erythrocytes and that a significant impairment of catalytic efficiency is also involved, as a result of the underlying structural mutation in the case of G6PD Ferrara II.

Adult↗
PubMed1981

The interference of leukocytes and platelets with measurement of clucose-6-phosphate dehydrogenase activity of erythrocytes with low activity variants of the enzyme.

Complete removal of leukocytes and platelets from whole blood showed that the glucose-6-phosphage dehydrogenase (G6PD) activity in "pure" erythrocytes from G6PD deficient hemizygous Sardinian subjects is consistently lower than reported in the literature. Thus, although non of 27 hemizygous subjects showed undetectable erythrocyte G6PD activity, their levels ranged between 0.0015 and 0.008 IU/g Hb, as compared with a mean value of 4.5 IU/g Hb in normal subjects. Most of the biochemical peoperties that were formerly ascribed to erythrocyte G6PD appear to be those of the enzyme from contaminating leukocytes and (or) platelets.

Antibodies↗
PubMed1981

Genetic variation in the quantitative levels of an NADP (H)-binding protein (FX) in human erythrocytes.

FX is a red cell NADP(H)-binding protein that has been well defined biochemically and immunologically but whose function is still unknown. Preliminary data indicated that the levels of this protein are significantly increased in hemizygotes, heterozygotes, and homozygotes for the G6PD Mediterranean mutant, thus raising the question of whether or not the individual variation in FX levels is more or less directly influenced by X-linked genes. The present study, based on a large series of population and family data collected in Sardinia, confirms unequivocally the above mentioned interaction, but shows at the same time that the variances in FX levels "between sibships" are 2-3 times larger than those "within sibships," when the analysis is done separately for the G6PD-normal or the G6PD-deficient sibs. From the comparison of the interclass and intraclass correlation coefficients, it appears that about 60% of the total variation of FX is of genetic origin. Moreover, the FX levels of children, analyzed in a pairwise manner, were found to be more positively correlated with those of their fathers (r = 0.39) than with those of their maternal grandfathers (0.20). This latter finding obviously favors the conclusion that "autosomal" rather than "X-linked" genes are involved in the determination of the FX levels.

Adult↗
PubMed1980

Identification of proteolytic activities in the cytosolic compartment of mature human erythrocytes.

Individual lysates from human erythrocyte suspensions, completely deprived of leucocytes and were assayed for a number of proteolytic activities using both naturally occurring and synthetic substrates. Removal of hemoglobin by batchwise DEAE-cellulose chromatography did not modify the complement of the various proteolytic activities which were then fractionated by means of chromatography on a column of DEAE-cellulose, followed by conventional techniques such as gel chromatography and preparative electrophoresis. This procedure allowed a number of proteinases to be identified in the erythrocyte cytosol while providing a tool for their selective though partial separation. The following peptidases were found to be present in the soluble fraction of mature human erythrocytes: (a) a neutral endopeptidase having an approximate molecular weight of 110 000; (b) three acidic endopeptidases, with pH optima between 2.5 and 3.5, showing molecular and functional properties almost identical with those of the three proteinases previously purified from solubilized erythrocyte membranes [Pontremoli et al. (1979) Biochem. J. 181, 559--568]; (c) two dipeptidylaminopeptidases whose molecular weights are around 80 000 and tentatively identified as dipeptidyl aminopeptidases II and III, respectively, on the basis of their substrate specificities and pH optima; (d) presumably two aminopeptidases, having an approximate molecular weight of 80 000 and classified as an aminopeptidase with broad substrate specificity and an aminopeptidase B, respectively. No evidence for any carboxypeptidase activity was found in the cytosolic compartment of mature human erythrocytes.

Cations, Divalent↗
PubMed1979

Isolation and partial characterization of three acidic proteinases in erythrocyte membranes.

1. The distribution of proteolytic activity in membranes from human erythrocytes and from rabbit reticulocytes and erythrocytes was investigated, after removal of leucocytes and platelets from the cell suspensions. 2. All membrane preparations displayed proteolytic activity in the acidic pH region only. Membranes from human and rabbit mature erythrocytes showed latent activity, which could be increased when extracted with a number of detergents. 3. Three active fractions were resolved either by gel chromatography of solubilized membrane extracts or by standard polyacrylamide-gel electrophoresis. The three proteinase activities (designated proteinases I, II and III) were purified from solubilized extracts of human erythrocyte membranes. 4. The relevant mol.wts. were around 80000, 40000 and 30000, respectively, and each of the three proteinases appeared to be composed of a single polypeptide chain. 5. Distinctive pH optima (in the range pH2.8-3.9) and different saturation profiles with globin as substrate were observed for proteinases I, II and III. 6. Dithioerythritol, Hg(2+) and Cu(2+) inhibited each of the three human enzymes, but more selective inhibitory effects were exerted by other modifiers of proteolytic enzymes and by haemin. Similar effects were observed with the three proteinases from rabbit cells. 7. The activity of the three human proteinases seems to be restricted to naturally occurring protein substrates, although with poor specificity, and none of them was active on synthetic substrates. 8. Digestion of globin by each of the three enzymes yielded similar polypeptide fragments in all cases, this indicating an endopeptidase type of activity.

Animals↗
PubMed1978

Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency.

A solid-phase radioimmunoassay for human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) was developed that allowed the specific activity of this enzyme protein to be measured in lysates from whole erythrocyte populations, in lysates from erythrocytes of different ages, and in purified samples. The enzyme was highly purified from erythrocytes of single donors by a simple procedure of affinity chromatography with insolubilized adenosine 2',5'-bisphosphate. These techniques were used in an attempt to elucidate the molecular mechanisms leading to deficiency of glucose-6-phosphate dehydrogenase activity in two genetic variants of the enzyme, i.e., the Mediterranean and the Seattle-like variants. The results indicate that the lowered activity of erythrocytes containing the Mediterranean variant of glucose-6-phosphate dehydrogenase is related to an enhanced rate of degradation of a catalytically defective protein synthesized at a nearly normal rate. Synthesis of a normally functioning protein and an increased breakdown of it are involved in the Seattle-like variant of the enzyme.

Cross Reactions↗