J. Sans

IMPAIRED CHROMOSOME SEGREGATION IN PLANT ANAPHASE AFTER MODERATE HYPOMETHYLATION OF DNA

10−6 M and 10−5 M 5‐azacytidine, demethylated around 9% and 17% of the 5‐methylcytosine residues found in Allium cepa L. native DNA, respectively. Both treatments stimulated RNA synthesis in the cells of root meristems. On the other hand, the 10−5 M treatment gave rise to multiple chromosomal anomalies in mitosis before any fall in the mitotic index was detectable, but no chromosomal breaks were ever seen.

Colchicine-resistant assembly of tubulin in plant mitosis

SummaryTubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 μm long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.

Cell proliferation in the mouse parotid gland after unilateral parotidectomy.

Unilateral parotidectomy with or without total submandibulectomy has been used to induce cell proliferation in mouse parotid gland. Maximum DNA synthesis and mitosis were recorded four and five days after the operation. The double operation increased the proliferative response. Such proliferative stimulii was not accompanied by secretion and was sex independent. On the other hand, the response decreased with age. RNA and protein synthesis inhibitors showed that the stimulation of DNA synthesis depends on early protein synthesis, which seems to be synthesized on a preexisting template.

Influence of copper-(II) on colloidal carbon-induced kupffer cell-dependent oxygen uptake in rat liver: Relation to hepatotoxicity

Formation of reactive O2 species in biological systems can be accomplished by copper-(II) (Cu2+) catalysis, with the consequent cytotoxic response. We have evaluated the influence of Cu2+ on the respiratory activity of Kupffer cells in the perfused liver after colloidal carbon infusion. Studies were carried out in untreated rats and in animals pretreated with the Kupffer cell inactivator gadolinium chloride (GdCl3) or with the metallothionein (MT) inducing agent zinc sulphate, and results were correlated with changes in liver sinusoidal efflux of lactate dehydrogenase (LDH) as an index of hepatotoxicity. In the concentration range of 0.1-1 microM, Cu2+ did not modify carbon phagocytosis by Kupffer cells, whereas the carbon-induced liver O2 uptake showed a sigmoidal-type kinetics with a half-maximal concentration of 0.23 microM. Carbon-induced O2 uptake occurred concomitantly with an increased LDH efflux, effects that were significantly correlated and abolished by GdCl3 pretreatment or by MT induction. It is hypothesized that Cu2+ increases Kupffer cell-dependent O2 utilization by promotion of the free radical processes related to the respiratory burst of activated liver macrophages, which may contribute to the concomitant development of hepatocellular injury.

Determination of the replication time of nucleolar organizer DNA after 5-azacytidine treatment for restricted parts of the S period

SummaryIn vivo exposure to 5-azacytidine (10−6M) depressed the incorporation of methyl groups to GC rich regions ofAllium cepa L. DNA. Nearly 22% of its 5-methylcytosine residues were under-methylated. The treatment stimulated 1.8 times the rate of [3H]uridine incorporation, as measured in meristems proliferating under steady state kinetics. Nucleologenesis was shortened from 2.7 to 1.6 h in synchronous binucleate cells after 5-azacytidine treatment lasting the whole S period of their previous interphase. By hypomethylating DNA sequences replicated at different times during the S period, it could be inferred that the cistron replication took place in early S. Thus, nucleogenesis was shortened to only 0.6 h after such treatment. Sequential short treatment periods using [3H]thymidine confirmed that the nucleolar organizer regions of the chromosomes replicate in early S.

Proliferative index estimated by chromatin pattern analysis

Chromatin pattern analysis at different density thresholds in Feulgen-stained meristems stimulated to proliferate by water inbibition, allows to estimate a proliferative potential index (PPI) which is a much earlier indicator of changes in proliferation than conventional labelling and mitotic indices. The PPI is but the ratio G1 cells to total 2C cells (or G2 to 4C cells, when cells also digress from the post-replicative stage of the cycle). The method is based in the fact that G1 and G2 cells have larger projected nuclear area and smaller dense chromatin area than their conterpart non-proliferating G0 and G0,2 cells.

Permeability changes induced by polylysines in rat spermatids

High molecular weight (HMW, >15 kDa) but not low molecular weight (LMW, <15 kDa) polylysines (PLs) bound and induced permeability changes in rat spermatid plasma membranes, estimated by Mn2+ quenching of intracellular indo‐1 fluorescence (K 1/2 = 3.3 ± 0.5 μg/ml) and Co2+ quenching of intracellular calcein. The pharmacology of the Mn2+ entry pathway activated by HMW PL does not suggest that Ca2+ channels are involved in this phenomenon. Concentrations of HMW PL that induced divalent ion entry did not induce the entry of ethidium bromide, suggesting that HMW PL first bound and perturbed the plasma membrane structure inducing a non‐specific increase in membrane permeability. High concentrations of HMW PL induced cell lysis (K 1/2 = 23 μg/ml). The binding of HMW PL, initially homogenous on the cell surface, subsequently progressed to a segregated pattern resembling a clustering phenomenon.

Early and late replicating DNA involved in the G1 to S transition in Allium cepa L meristematic cells

Summary— The involvement of portions of the genome replicated at different times of the S period in the regulation of the G1 to S transition was analyzed in Allium cepa L meristem cells. For this, DNA bromosubstitution confined to discrete portions of a previous S period followed by anoxic UVA irradiation (300–400 nm light) was performed in synchronous cells. Sequences replicated in late S appeared to be involved in the positive regulation of the initiation of replication. Hence, cells were prevented from initiating replication if irradiated at mid G1 only when the DNA sequences replicated in the last third of the previous S period were bromosubstituted. Cycloheximide‐induced inhibition of protein synthesis at late G1 also prevented the G1 to S transition. Sequences replicated in mid S appeared unrelated to any control of the initiation of replication. On the other hand, sequences replicated in the first third of the S period seemed to be involved in the negative regulation of the initiation of replication, since irradiation after previous bromosubstitution of early replicating DNA sequences advanced G1 cells into the next S phase and increased the proliferative fraction of the population. Finally, the simultaneous inactivation of DNA sequences involved in both positive and negative regulation of replication allowed the cells to enter into S.

Cis-acting loci involved in the induction and reversal of chromosome condensation in plant prophase, determined by their different replication times

SummaryAnoxic UVA irradiation (300–400 nm) of cells in prophase induced their chromatin to return to the interphase decondensed form when their DNA was unifiliarly bromosubstituted. This immediate effect may be related to the incompetence of chromatin with Br-DNA when irradiated to bind proteins which induce its condensation. Hence, inhibition of protein synthesis also causes chromatin decondensation in cells with native DNA.Bromosubstitution of DNA sequences replicated in the last two thirds of the S period was as efficient as bromosubstitution of the whole genome for such an effect to take place in a nucleus. On the other hand, the irradiation accelerated the entrance into prophase of those cells in which only sequences replicated in the first third of S were bromosubstituted. Thus, early replicating loci may act as attachment sites for binding proteins preventing the induction of chromatin condensation. DNA bromosubstitution during portions of S was carried out in synchronous cell populations labelled as binucleate by a previous short caffeine treatment, inAllium cepa L. root meristems.

The maintenance of colchicine‐arrested metaphases in plants requires protein synthesis

Abstract With the aim of finding out factors involved in chromosomal condensation, the kinetics of both metaphase accumulation and the subsequent formation of restitution nuclei in the presence of 1 mM colchicine have been analysed in Allium cepa L. meristems. Restitution nuclei are formed by decondensation of the chromosomes from the c‐mitoses and the reformation of nuclear envelopes around them. The study has been carried out in control conditions as well as in conditions which either inhibit protein synthesis (1 μg/ml cycloheximide) or modify the accuracy of transcription (by near ultraviolet light irradiation under anoxia of cells with bromosubstituted‐DNA).