Development of a nuclear polyhedrosis in cells of Rhynchosciara angelae (Diptera, Sciaridae) and patterns of DNA synthesis in the infected cells

An ultrastructural and autoradiographic study of the infection of cells of Rhynchosciara angelae by a nuclear polyhedrosis virus (RPV) is presented. RPV is a DNA virus and causes a dramatic increase in the volume of the infected cells and in the sizes of chromosomes and their DNA contents. The structure of the nucleoli changes with the infection and the changes are mainly related to an increase of DNA synthesis. The concentration of ribosomes increases in the cytoplasm of the infected cells. Autoradiographic study of the DNA synthesis showed that it varies with the infective process.Four patterns of DNA synthesis, in relation to the host chromosomes and the virus, were disclosed by means of tritiated thymidine incorporation in the infected nuclei. The patterns are: (1) incorporation mainly in the chromosomes, (2) incorporation in the chromosomes and in the nucleoplasm, (3) incorporation only in the nucleoplasm, and (4) incorporation mainly in the chromosomes in dissociation. There is indication of a succession 1→2 and 3→4. The succession of patterns indicates that the virus induces first the increase of synthesis of host cell DNA and RNA. The bulk of the synthesis of viral DNA is evident only after the host cell DNA and RNA machinery is amplified. The aspects of the formation of viral membrane indicate that it is a de novo process in which the membrane material is capable of self-assembly.     

Action of hydroxyurea on chromosome physiology in Rhynchosciara angelae

Experiments have been performed to investigate the action of hydroxyurea (H.U.) on the polytene chromosomes of the salivary gland of Rhynchosciara angelae. After different times of H.U. treatment, larvae were injected with ³H-thymidine for a pulse of 10 min. DNA puffs were analysed especially in those regions where differential incorporation of thymidine occurs. H.U. progressively inhibited thymidine incorporation all over the chromosome. The maximum of inhibition occurs 9 hours after the treatment. However, after 227 hours the chromosome label was similar to that in the controls and puff 2B recovered its original size. The puff 3C showed a delay in its appearance. Our results show that H.U. inhibits temporarily the opening rate of the puff, as well as DNA synthesis. There is no reaction on RNA puffs.This work was supported by a grant from the National Institutes of Health (GM 17590-03), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) of which one of us (G.M.M.S.) was a fellow during this research, and the Conselho Nacional de Pesquisas (CNPq).     

The rate of DNA replication in the polytene chromosomes of Rhynchosciara angelae

The distribution pattern of 5-bromodeoxyuridine-labelled DNA from salivary glands of Rhynchosciara angelae upon caesium chloride gradient centrifugation was studied with DNA of different molecular weights. This pattern suggests a very low rate of DNA chain growth in polytene chromosomes. The rate of DNA chain growth was found to be 0.025 μm/min at 24 °C. The result was obtained through the development of a mathematical expression which took into account the distribution of the 5-bromodeoxyuridine-labelled DNA in CsCl gradients.DNA pulse-labelled for a short time sediments more slowly in alkaline sucrose gradients than DNA which has been labelled during a prolonged incubation. However, in neutral sucrose gradients the pattern of banding is the same for both DNAs. This indicates a discontinuity in the newly synthesized DNA strand, but not in the template strand. The transition of slow sedimenting to fast sedimenting DNA observed in alkaline sucrose gradients, occurs very slowly, as would be expected for a slow rate of DNA chain growth.The data obtained provided a means of comparing the number of replication points with the rate of DNA chain elongation and the length of S phase in Rhynchosciara.     

Nucleic acid synthesis in preimplantation mouse embryos

Summary Mouse embryos were collected at the 2-cell stage, cultured in vitro in the presence of³H deoxyuridine or uridine for 6 or 4 h and autoradiographed.Deoxyuridine is actively incorporated into the DNA of cleaving mouse embryos indicating the existence of thymidylate synthetase activity at least at the 4-cell stage and presumably already before this.RNAase treatment of embryos squashed on slides shows a weak but obvious incorporation of uridine into DNA of cleaving mouse embryos, from the 4-cell stage onwards; this incorporation is totally inhibited by hydroxyurea. The reduction of ribonucleotides to deoxyribonucleotides is a metabolic pathway already required for cleavage, as shown by hydroxyurea experiments.The second polar pody, known to incorporate thymidine, is unable to incorporate either deoxyuridine or uridine.     

Nucleic acid synthesis in cancerous cells under the effect of gnidilatimonoein from Daphne mucronata

Cytotoxicity evaluation of gnidilatimonoein, the most active isolated diterpene ester from Daphne mucronata [Sadeghi H, Mianabadi M, Yazdanparast R, (2002) Journal of Tropical. Medicinal Plant1 3: 169-173], revealed the strong antiproliferative activity among several different human cancer cell lines (K562, CCRF-CEM, HL-60 and MOLT-4 leukemia cell lines, LNCaP-FGC-10 a prostate cancer cell line) and a mouse BALB/C fibrosarcoma cell line (WEHI-164). Using flow cytometry technique, it was found that treatment of the most responsive cells (K562) with gnidilatimonoein inhibited the progression of cells through G1 phase by almost 15% compared to the untreated cells. The population of the treated cells in the S and G2 phases also reduced by 8.3% and 5.4%, respectively. Based on the extent of [3H]-thymidine and [3H]-uridine incorporation into DNA and RNA, respectively, the major metabolic effects of gnidilatimonoein were found to be mainly on DNA and to a less extent on RNA synthesis. Additionally, the activity of inosine-5'-monophosphate dehydrogenase (IMPDH), under the effects of genidilatimonoein, was reduced in the treated cells by 44%. These data strongly suggest that the purine biosynthetic pathway is significantly affected by gnidilatimonoein.