Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

Effects of fluazifop-butyl {(±)-butyl 2-[4-[(5-(trifluoromethyl)-2-pyridinyl)oxy] phenoxy] propanoate} on cellular uptake and incorporation of 14C-leucine, 14C-uridine, and 14C-thymidine into protein, RNA, and DNA, respectively, were evaluated in corn (Zea mays L.) coleoptiles and soybean [Glycine max (L.) Merr.] hypocotyls. Uptake of 14C-leucine in corn coleoptiles was inhibited by fluazifop-butyl at 10-4 to 10-7 M, which was related to reductions in incorporation of 14C-leucine into protein at 10-4 to 10-6 M. The effect of fluazifop-butyl on 14C-leucine uptake and incorporation was reduced by 2 × 10-6 M 2,4-D [(2,4-dichlorophenoxy)acetic acid], indicating antagonism between these herbicides. RNA and DNA synthesis were inhibited at 10-5 and 10-4 M. Protein synthesis in soybean hypocotyls was stimulated by 10-7 and 10-6 M fluazifop-butyl 33 and 27%, respectively, and inhibited by 10-4 M 50%. RNA and DNA synthesis were also inhibited by 10-4 M in soybean.

The mode of action of two experimental pyridinyloxyphenoxy propionate herbicides, haloxyfop {2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy] phenoxy] propanoic acid} and CGA-82725 {2-propynyl-2-[4-[(3,5-dichloro-2-pyridinyl)oxy] phenoxy] propanoic acid} was studied. Oat (Avena sativa L.) roots showed a rate-dependent sensitivity to both herbicides. Consequently, a series of assays was performed to study cell division, cell cycle dynamics, and nucleic acid and protein synthesis in oat root tips in order to characterize growth inhibition. Both herbicides inhibited cell division, apparently by inhibiting protein synthesis in the G2 stage of interphase. Supporting evidence for this assumption comes from the following observations: a) There was almost complete absence of leucine incorporation within 1 h of exposure to 2.7 × 10−4 M of either herbicide; b) the number of 3H-labeled dividing cells was reduced to about 20% of the control within 4 h, and essentially to zero within 8 h, after exposure to 2.7 × 10−4 M of herbicide; c) the number of unlabeled cells was reduced to less than 10% of the control within 8 h of exposure to 2.7 × 10−4 M of herbicide; d) during this 8-h period, the number of 3H-labeled cells in interphase which had been exposed to herbicide greatly exceeded the control; e) thymidine incorporation into DNA was inhibited but was not proportional to reduction in cell division; f) uridine incorporation was enhanced during the first 8 h of exposure before a reduction was evident at 12 h; and g) an oat coleoptile elongation bioassay showed that cell enlargement was not inhibited at herbicide concentrations less than 2.7 × 10−4 M.

A large body of evidence has demonstrated that normal human fibroblasts have a limited division potential in culture and underwent senescence, a process whereby cells became arrested in the G1 phase of the cell cycle and overexpressed a DNA synthesis inhibitor(s). Cyclin-dependent kinase two (Cdk2) is required for the promotion of the Gi-to-S phase transition in human cells. Senescent fibroblasts contain intact cyclin-Cdk2 complexes but cannot induce Cdk2 protein kinase activity in response to mitogen stimulation. Recently, we cloned p21Sdi1, a potent inhibitor of DNA synthesis and Cdk2 kinase activity, from a senescent cell cDNA library and demonstrated that it was expressed at significantly higher levels in senescent cells than actively proliferating cells. In contrast to actively dividing cells, mitogen-stimulated senescent cells do not down-regulate the expression of p21Sdi1 and do not express late G1 phase gene products that are required for entry into S phase. We suggest that the inability of mitogen-stimulated senescent cells to down-regulate p21Sdi1 levels contributes to the resulting lack of late Gi gene expression and failure to traverse the G1/S phase boundary.

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.

We previously showed in organ culture of testes fragments from Cynops pyrrhogaster that mammalian folicle-stimulating hormone (FSH) stimulates secondary spermatogonia to differentiate into primary spermatocytes. In this report, we demonstrate in organ culture that FSH stimulates DNA synthesis in secondary spermatogonia and Sertoli cells: the numbers of secondary spermatogonia and Sertoli cells incorporating 5-bromo-2′ -deoxyuridine (BrdU)throughout the culture period in the presence of FSH were 3-5 times those incorporating BrdU in the absence of FSH. Moreover, addition of FSH, induced after a day a remarkable increase in the number of spermatogonia and Sertoli cells incorporating BrdU. The above results indicate that FSH stimulates and induces DNA synthesis in spermatogonia and Sertoli cells. Most of the spermatogonia within a cyst were labelled simultaneously and at the same density, indicating that they underwent synchronous DNA synthesis, whereas all the Sertoli cells within a cyst were not labelled simultaneously, indicating that they synthesised DNA asynchronously. When testes fragments pulse-labelled with Brdu were cultured in FSH for 14 days, the secondary spermatogonia differentiated into primary spermatocytes, whereas in the absence of FSH they failed to differentiate and most died by the 7th day. The above results together show that FSH is required for the proliferation of both secondary spermatogonia and Sertoli cells as well as the differentiation of secondary spermatogonia into primary spermatocytes.

Mouse, sheep and bovine metaphase II oocytes were briefly preincubated in medium with 6-dimethyl-aminopurine (6-DMAP) and thereafter activated with ethanol before further culture in 6-DMAP supplemented medium. The presence of 6-DMAP enhances the efficiency of activation and the speed of pronuclear formation but has no effect on DNA synthesis. These results are discussed in the context of recently published data showing that the preincubation of condensed chromosomes with 6-DMAP blocks DNA synthesis when nuclei become reformed.

Parthenogenetically activated mammalian oocytes have been used in the past decade as cytoplasts, in an attempt to support the development of nuclear transplant embryos. The present experiments were undertaken to study the DNA synthesis and the organisation of microtubules, nuclear envelope and chromatin during the first cell cycle of electrically activated porcine oocytes (parthenotes) matured in vitro by using immunocytochemistry and laser scanning confocal microscopy. The results showed that pronuclear-like (PN) formation began 4–5 h post-activation (hpa), whilst DNA synthesis as revealed by bromodeoxyuridine incorporation was initiated 5–6 hpa, with a maximum number of labelled oocytes (73%) around 11 hpa, and persisted in some parthenotes until 15–16 hpa. In the metaphase II (MII) oocytes, microtubules were detected only in the metaphase II spindle; no lamin A/C antigen was observed. Electrical DC pulses resulted in 91% of MII oocytes being activated and confocal microscopy Indicated that microtubules were assembled in the spindle first for the extrusion of a second polar body, and for the second time for division from one to two cells. Nuclear envelope, indicated by anti-lamin A/C stain, was formed around the time of PN formation and surrounded the nuclear chromatin of 1- and 2-cell parthenogenotea. These results demonstrate that the apparent normality in both DNA synthesis and dynamics of microtubules and nuclear envelope is involved with chromosomal organisation in the parthenotes. In addition, the use of electrically activated IVM oocytes for both nuclear transfer and parthenogenetic studies in pigs is discussed.

Sugarbeet (Beta vulgaris) seeds which had undergone an advancement treatment germinated more quickly and showed greater germination in a standard test at 9°C than seeds which had been steeped in water. Both treatments included the fungicide, thiram (tetramethyl thiuram disulphide). Advanced seeds also emerged more quickly and showed higher establishment in the field. The levels of extractable high molecular weight (HMW) RNA and DNA from advanced, thiram-steeped and untreated seeds were compared. Advanced seeds contained significantly (P > 0.05) more HMW RNA and DNA than thiram-steeped or untreated seeds. Extractable DNA levels were higher from thiram-steeped seeds than untreated seeds but not significantly so. Cell-cycle studies on advanced seeds demonstrated an increase from 2% to 24% of root-tip nuclei with DNA contents of 4C and above. This indicates that DNA replication occurred during the advancement treatment. There was no evidence of cell division occurring, indicating that: (1) advanced seeds were arrested post-S-phase and (2) the period following DNA synthesis in sugarbeet seeds is desiccation tolerant.

The conditions of osmopriming of maize seeds have been established. At an osmotic potential of–1.7 MPa, seed germination is inhibited and seeds osmoprimed for up to 3 weeks show an improved germinability when the osmotic agent is removed. This improvement also applies to seeds that lost vigour because of improper storage conditions. At the biochemical level, embryo axes from osmoprimed seeds can incorporate precursors into DNA, RNA and proteins although at a low level; after removing the osmotic agent, all 3 types of macromolecules are synthesized at much higher levels. No evidence of DNA replication or cell division was found during osmopriming; however, mitotic figures appear several hours earlier in germinated, osmoprimed root tissues compared with the time at which they appear in non-osmoprimed tissues. The behaviour of osmoprimed maize is compared with that of osmoprimed seeds from other plant species.

The effect of natural and artificial seed ageing has been compared in terms of physiological and biochemical responses of several maize genotypes. The physiological parameters were: viability, germinability, emergence in sand and dry matter accumulation. The biochemical parameters were: DNA synthesis and DNA polymerase activity. A close and direct relationship was found between seed deterioration and DNA metabolism in all maize materials which responded to ageing according to their genetic constitution; i.e., vigorous genotypes suffered less severe damage or recovery was faster than in the low-vigour genotypes. Coordination of events at appropriate times would seem a critical factor for proper seed germination.

We have studied by means of cytology and autoradiography the effect of benzyladenine (BA, a synthetic cytokinin) on DNA metabolism during early maize germination.The data indicate that BA stimulates nuclear DNA replication. The doubling of the amount of nuclear DNA in BA-treated axes occurs earlier than in nontreated axes, and there is a three-fold increase in the mitotic index at 24 h of germination. These results provide further corroboration for the suggestion that the stimulation of DNA synthesis observed relates to a nuclear replicative type of synthesis. Possible mechanisms of action of BA are discussed.

Cell cycle activities are initiated following imbibition of non-dormant seeds. However, it is not known whether cell cycle related events other than DNA replication also remain suppressed in imbibed dormant seeds. The objective of this study was to demonstrate that the transitions between the non-dormant and dormant (both primary and secondary) states are reflected in cell cycle events, such as DNA replication and the changing patterns of the microtubular cytoskeleton involved in the processes of growth and development. The present studies were conducted on seeds from tomato (Lycopersicon esculentum cv. Moneymaker) that possessed primary dormancy or were manipulated to attain secondary dormancy. In addition, a non-dormant abscisic acid (ABA)-deficient mutant, sitw, was used. DNA replication, as measured by flow cytometry, and β-tubulin accumulation, analysed by immunoblotting, were compared with immunocytological studies of active DNA synthesis and microtubular cytoskeleton formation. It is shown that the depth of dormancy, which distinguishes primary and secondary dormancy, may depend on the progression of the cell cycle prior to the induction of dormancy.

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 μCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 μCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of ≤ 1 h, 22% had a G1-phase of ≤ 2 h, 22% had a G1-phase of ≤ 3 h and 13% had a G1-phase of ≤ 4 h. The G1-phase in 85% of the 4-cell embryos was ≤ 3 h, that in 8% of embryos was ≤ 4 h and that in 7% of embryos was ≤ 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 μM, and the appropriate concentrations which did not impair development were 0.05-0.5 μM.