Biomedical subjects
Publications and source records attributed to F Nuzzo.
We analysed by liquid scintillation counting and by autoradiography the DNA repair synthesis induced by UV irradiation in mononucleated cells from peripheral blood of 'hairy cell leukaemia' patients. In 9 out of 12 patients the repair activity values obtained in repeated assays were found to be significantly lower than those of healthy donors.
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The variation of DNA repair activity during the cell cycle was studied by analysing the UV-stimulated DNA synthesis in cells synchronized in mitosis. This activity was detected both by autoradiography and by directly measuring the incorporation of tritiated thymidine in cells irradiated and incubated in the presence of hydroxyurea. Cells in all phases were found to be able to perform repair. However the activity appeared to be considerably lower in mitotic cells than in cell in other phases. Increasing values of repair capacity were observed in G1 cells, in mixed G2, S and M cells and in asynchronous cells. The relationship between these findings and data on survival rates in the same synchronized cells is discussed.
Two cases of reciprocal translocation between chromosomes 3 and 4 found in unrelated families are described. The meiotic consequences of this rearrangement are discussed.
We have determined the levels of DNA-polymerases-alpha and-beta in fibroblasts obtained from normal subjects and from patients with Xeroderma Pigmentosum (XP) belonging to three different complementation groups and to the variant form. The assays have been performed in crude extracts and after fractionation on sucrose gradients. The levels of alpha and beta-polymerases in the different cases of XP were found to lie within the same range as the control values, and no correlation was found with the severity of the symptoms. The sedimentation coefficients of the two polymerases from all the pathological lines were identical to those of the normal fibroblasts.
We studied DNA repair synthesis after ultraviolet irradiation in human fibroblasts cultured in vitro by measuring the ultraviolet-stimulated incorporation of [3H]thymidine into cells in which the semi-conservative DNA replication was inhibited by hydroxyurea. Experiments performed with five fibroblasts lines derived from healthy donors showed a relatively fast initial process ( that is completed within 1 h for 100 erg/mm2 and within 2 h for 500 erg/mm2) and a subsequent slower process, evident between 2 and 6 h after irradiation. The repair capacity of normal cells is expressed by the difference between the values of incorporation (in presence of hydroxyurea) of irradiated and control cells. The pattern of repair was similar in all five cell lines: repair capacity was positive and the amount of repair synthesis increased with incubation time after UV irratiation. Similar experiments were performed with fibroblasts derived from five patients with the classical xeroderma pigmentosum (XP) and from one patient with the De Sanctis-Cacchione syndrome. Normal and XP cells could be distinguished according to whether they displayed a positive or negative value of repair synthesis and/or according to the degree of the slope of the repair synthesis curve as a function of the incubation time after irradiation. We conclude that the technique used in our experiments can demonstrate in a rapid and simple way a defect in the repair capacity in fibroblast cultures; the data are in good agreement with those obtained in the same XP cell lines by other authors [9], who have measured unscheduled DNA synthesis in autoradiographs and repair replication after addition of BUdR.
Stimulation of human lymphocytes with phytohemagglutinin is known to induce an increase in overall DNA polymerase activity (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). Previous work [Pedrali Noy, G., Dalprà, L. Pedrini, A. M., Ciarrocchi, G., Giulotto, E., Nuzzo, F. & Falaschi, A. (1974) Nucleic Acids Res. 1, 1183] has shown that two subsequent waves of induction of DNA polymerase can be observed in this system; a first wave occurs in parallel with the increase in DNA replication rate; a second one occurs when the DNA synthesis rate is returned to minimal levels; the second peak is parallel to a maximum in DNA ligase and DNase levels. In the present work we have measured the levels of the DNA polymerases-alpha and -beta in phytohemagglutinin-stimulated lymphocytes during a 12-day period; both enzymes are present at detectable levels at time zero; in correspondence to the peak of DNA synthesis rate (between the fourth and fifth day) a peak of DNA polymerase-alpha is observed, increasing by a factor of approximately 20-fold over the zero time value; subsequently, the level of DNA polymerase-alpha decreases in parallel with DNA synthesis rate. The DNA polymerase-beta is also increased in correspondence to the peak in DNA synthesis rate, but reaches its maximum at later times, between the eighth and tenth day of incubation. The capacity of stimulated lymphocytes to perform repair synthesis following UV damage was measured in the same cells used for the enzyme activity determinations; this capacity also shows two maxima: a first one correlated with the peak in DNA replication rate, and a second one correlated with the peak of DNA polymerase-beta. These data suggest a certain tendency to the specialization of functions in human cell DNA polymerases; the alpha-enzyme seems mainly correlated with DNA replication, whereas the beta-enzyme seems more correlated with the ability of the cell to perform repair type synthesis.
Karyotype analysis was performed on successive cultures of fibroblasts from the parents of two trisomic 21 patients. Starting from the 7th passage in the father cultures an aneuploid clone showing an extra E-like chromosome was found, which eventually overgrew the cell population. The significance of this cytogenetic finding is discussed in relation to the recurrence of the trisomy in the family.
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A family with three sibs, including a pair of dizygotic twins, all affected by Down's syndrome with regular trisomy 21, is described. The chromosome counts carried out on prolonged fibroblasts cultures of the mother, revealed the presence of the trisomy 21 in 6 out of 688 scored mitoses. The cytological findings give support to the hypothesis of a chromosome mosaicism in one of the normal parents, as a cause of the recurrence of the trisomy 21.
We have determined the levels of DNA polymerase, DNA ligase, a DNase acting on single-stranded DNA, an endonuclease making single-strand breaks in double - stranded DNA and polynucleotide kinase in fibroblasts obtained from nine normal persons and from nine patients with Xeroderma Pigmentosum; the pathological lines belong to the different described clinical forms and to the three different complementation groups described so far. All the enzymes are present in the normal lines and in the Xeroderma lines. The levels are quite variable, but the values obtained in the pathological lines lie within the ones observed in the normal population.
The stimulation of human lymphocytes with phytohaemoagglutinin induces the appearance or increase of several enzymes of DNA metabolism [Pedrini etal., Biochem. Biophys. Res. Comm., 47:1221(1972)]. With long times of stimulation, two phenomena are observed; an increase in the levels of DNA polymerase, of a DNase acting on single-stranded DNA, and of an endonuclease, occurring between the third and fourth day, in parallel with a wave of DNA synthesis;a second wave of increase of the same enzymes and of DNA ligase,occurring between the fifth and eight day when the DNA replication rate, as measured by thymidine-pulses, has decreased to values close to the background.
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The newly synthesized DNA of heteroploid human cells cultured in vitro has a single-stranded molecular weight of approximately 1 x 10(5) following 30- to 60-second pulses of labeling with tritiated thymidine. With increasing pulse times the amount of radioactive DNA having a low single-stranded molecular weight remains approximately constant, whereas increasing amounts of label are found associated with the bulk DNA. After removing the label from the medium, the radioactivity associated with the small molecular weight DNA is chased into high molecular weight material. These findings are analogous to what is observed in microorganisms and suggest a basically common organization of the growing point of DNA in bacterial and in mammalian cells.
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