pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract.

In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10. In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs. We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10. The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation. Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts. These data identify pEg2 as a good candidate for mitotic histone H3 kinase. However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs. This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae.

Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex binding.

Here we have examined HIV-1 nucleosome remodeling upon the binding of transcription factors and the SWI/SNF complex using a novel approach. The approach combines UV laser protein-DNA crosslinking, electrophoretic mobility-shift analysis and DNase I protection analysis with immunochemical techniques. It was found that single activator-bound HIV-1 nucleosomes exhibit very weak perturbation in histone NH(2) tail-DNA interactions. However, the simultaneous binding of the transcription activators Sp1, NF-kB1, LEF-1 and USF synergistically increased the release of histone NH(2) tails from nucleosomal DNA. In contrast, the binding of SWI/SNF complex to HIV-1 nucleosome disrupted structured histone domain-DNA contacts, but not histone NH(2) tail-DNA interactions. Stable remodeled nucleosomes, (obtained after detachment of SWI/SNF), displayed identical structural alterations with those bound to SWI/SNF. These results demonstrate a different in vitro remodeling of the HIV-1 nucleosome upon the binding of multiple transcription activators and of SWI/SNF complex.

[In vitro stimulation by ellipticine derivatives of DNA cleavage induced by DNA topoisomerase II: a structure-activity relationship]. / Stimulation in vitro par des dérivés de l'ellipticine des coupures de l'ADN induites par l'ADN topo-isomérase II: relation structure-activité.

Ellipticines are intercalating planar polycyclic aromatic molecules that display antitumor activity. The cytotoxicity of these compounds is related to the presence of an hydroxy group at position 9 of the pyridocarbazole ring system and to their interaction with DNA topoisomerase II. The ability of 13 ellipticine derivatives to stabilize the topoisomerase II-DNA covalent complex in vitro is reported. The following observations emerge from our structure-activity relationship study: i) the hydroxy group at position 9 is essential for stabilizing the covalent complex, ii) the replacement of the methyl group at position 5 by an ethyl group (EPC) enhances the complex stabilization. The interaction of EPC and three other ellipticine analogues with DNA shows that the covalent complexes which are most stable have the lowest drug-DNA binding constants. In addition our study suggests that ellipticines induce covalent complex stabilization by a cooperative mechanism. A model is proposed to explain this stabilization by ellipticines. This study supports the idea that topoisomerase II is the primary target involved in the mechanisms of action of ellipticines.

Stimulation of topoisomerase II-mediated DNA cleavage by ellipticine derivatives: structure-activity relationship.

Ellipticines are aromatic compounds that intercalate between DNA base pairs and display significant antitumor activity. The cytotoxicity of these compounds is mediated by DNA topoisomerase II, and the presence of a hydroxy group at position 9 of the pyridocarbazole ring system of ellipticines has been found to be essential for high levels of cytotoxicity. The ability of 13 ellipticine derivatives to stabilize the topoisomerase II-DNA covalent complex in vitro was studied, and the data obtained with five pairs of hydroxylated and nonhydroxylated analogues indicate that the hydroxy group at position 9 plays a crucial role in the stabilization of the complex. The influence, upon the complex stabilization, of various substituents at positions 1, 2, 5, and 6 of the pyridocarbazole ring system was investigated. The interaction with DNA of four ellipticine derivatives was studied in the topoisomerase II standard medium. Results suggest that the degree of unwinding might be a determinant of topoisomerase II-DNA-drug complex stability. In addition, the 5-ethyl derivative was observed to induce covalent complex stabilization by a cooperative mechanism.

DNA-drug recognition and effects on topoisomerase II-mediated cytotoxicity. A three-mode binding model for ellipticine derivatives.

Cytotoxic effects and topoisomerase II-mediated DNA breaks induced in vitro by ellipticine derivatives were examined in connection with 1H NMR and circular dichroism (CD) studies on molecular structures and interactions of drugs with DNA. The compounds included four 9-hydroxyellipticine and two 7-hydroxyisoellipticine derivatives. Structure-activity relationships indicated that a change in nitrogen atom position in the pyridinic ring greatly affected drug effects both on topoisomerase II action and cytotoxicity to L1210 cells. The four 9-hydroxyellipticine derivatives yielded bell-shaped curves in in vitro topoisomerase II-mediated DNA break assays, whereas the two 7-hydroxyisoellipticine derivatives demonstrated an almost linear increase at the same concentration (0-10 microM). In both cases, the intensity of cleavage was modulated by the position and the degree of methylation on the pyridinic ring, and results were correlated with cytotoxic activity expressed as the in vitro ID50 values for L1210 leukemia cells. 1H NMR experiments performed on free drug molecules in solution revealed that the two protons (alpha and beta) contiguous to the biologically important hydroxy group were sensitive to changes in electron distribution produced by the distant chemical modifications and methylations of the pyridinic ring. A linear relationship was observed between the differences in chemical shifts of alpha and beta protons (delta delta alpha-beta) versus ID50 values. CD experiments indicated that, at weak ionic strength I = 0.02 and at pH 7, drugs interact with the poly[d(A-T)] duplex according to a "three-mode binding model" which is governed by the drug structure and the drug to DNA ratio. The intercalation mode was related to the induction of topoisomerase II-mediated DNA cleavage, while the external binding mode consecutive to intercalation was related to cleavage suppression. These two modes concerned the good intercalators 9-hydroxyellipticines. The third was found for the weak intercalators 7-hydroxyisoellipticines and was characterized by self-stacked molecules bound "outside" DNA, presumably in the minor groove. Ligands either could be intercalated partially or linked at the edge of bases with a small number of molecules filling intercalation sites, for the second alternative. In addition to having different binding modes, 9-hydroxyellipticines were better inducers of DNA distortions than 7-hydroxyisoellipticines. The incidence of the drug binding modes on DNA-topoisomerase II recognition was discussed in connection with the in vitro cytotoxic activity exhibited by the drugs.

Deficient natural killer cell activity in alcoholic cirrhosis.

Natural killer (NK) cell activity against two types of target cells was found to be low in patients with inactive alcoholic cirrhosis (AC). This defect was significantly more pronounced in AC patients with severe malnutrition than in those with mild or moderate malnutrition. This was not due to modifications of the kinetics of NK activity. The sera from AC patients had no inhibitory effect on the NK activity of normal subjects. Lymphocytes and macrophages from AC patients did not exert major suppressive effect on the NK activity of normal subjects. Interferon boosted the NK activity of cells from AC patients, but to a lesser degree than cells from normal controls. The findings show that a deficit of NK activity is clearly associated with inactive AC. This seems to be another consequence of AC on cellular immunity, and might be related to the protein calorie malnutrition often present in AC.

Nutrition and immunity after peritoneovenous drainage of intractable ascites in cirrhotic patients.

Severe malnutrition and related impaired cell-mediated immunity are commonly found in cirrhotic patients with intractable ascites and may be responsible for the poor prognosis of these patients. The effects of LeVeen peritoneovenous shunting of ascites on protein reserves and cell-mediated immunity were studied in 31 cirrhotic patients with intractable ascites over a period of 1 year following operation. Arm-muscle circumference, serum albumin, and transferrin levels markedly increased and became close to the normal values. In the same period, delayed hypersensitivity improved. Although the lymphocyte count and the absolute T cell concentration in peripheral blood remained low after peritoneovenous shunting, the in vitro lymphoblastic transformation in response to phytohemagglutinin was augmented. An increased capacity to eat normal meals and the resumption of a good appetite due to the discontinuation of a salt-restricted diet seemed to be the most important factors in the dramatic improvement in the nutritional status of these patients. Restriction from heavy alcohol use may have also contributed, although the patients had already stopped drinking for several months before operation. Improvement in cell-mediated immunity might have been secondary to the increase in protein reserves.