The effect of adenosine analogue (DRB) on a major nucleolar phosphoprotein nucleolin.

Nucleolin, a phosphorylated nucleolar protein, of 100 kDa selectively stained with bismuth tartrate and silver nitrate, is implicated in the transcription and maturation of pre-ribosomal RNA. Nucleolin also fulfills a structural function in nucleolar organization. Using immunocytochemistry the action of 5-6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of hn/RNA synthesis known to modify the organization of the nucleolus, was studied for its effects on the distribution and the amount of nucleolin present. After DRB treatment, the morphology of the nucleolus was rapidly disturbed, but the distribution of the nucleolin remained unchanged: the dense fibrillar and the granular components were always positively immunostained. Thirty min after incubation with the drug, a strong increase of the amount of nucleolin occurred. Prolonged treatment led to a marked loss of label. Silver and bismuth staining showed that DRB does not seem to significantly affect the phosphorylation of nucleolin.

Correlation between rDNA transcription and distribution of a 100 kD nucleolar protein in CHO cells.

A major nucleolar protein with a molecular weight of 100 kD is directly implicated in the transcription of pre-ribosomal RNA (pre-rRNA) and appears to be cleaved into specific maturation products during pre-ribosome biogenesis. Polyclonal antibodies which recognize the 100 kD protein and its products were used to determine the correlation between rDNA transcription and these proteins. Actinomycin D (AMD) was used to block selectively rDNA transcription (AMD 0.1 microgram/ml). Immunoperoxidase and immunogold staining were carried out in untreated and treated cells. Digitalization allowed the quantification of label according to the nucleolar components and the cellular areas. In exponentially growing cells, the dense fibrillar component was shown to contain more 100 kD protein than the granular RNP component but both nucleolar components were positively immunostained. The distribution of the 100 kD protein was rapidly modified by AMD: loss of label occurred first in the dense fibrillar zone of the nucleolus, demonstrating the correlation between rDNA transcription and the presence of this protein. However, one part of the protein remains in the segregated nucleolus after 1 h of AMD treatment, thus supporting the structural function of this protein.

Study of a ternary diagram gel according to a methodology of experimental research.

Synopsis The purpose of this work was to compare two methods to determine the formulation of a gel: a 'classical'method previously used and a rational method using matrixes based on the Scheffe algorithm which is particularly useful in the study of these type of mixtures. This gel was composed of three products: C12-C15 Alcohols Benzoate, Cyclomethicone and Ethylene Vinyl Acetate Copolymer in water. The ratios of these compounds were studied according to five criteria: compatibility between phases, consistency, whiteness, cost and 'skin spreading'. To determine the incompatibility zone inside the ternary diagram, we carried out our study by a systematic sequential walk strategy (seven experiments). Three parameters (consistency, whiteness, cost) were optimized using the Scheffe algorithm. This method only needs a limited number of experiments on which to base an empirical mathematical model of the studied phenomena as a function of the different experimental factors. The formulation is therefore discussed as a function of the first degree linear, the second degree and a third degree 'reduced cubical'model. The validity of the possible models are also discussed and the 'reduced cubical'best fits our phenomena.

Effect of pH and monovalent cations on the ionization state of phosphatidylglycerol in monolayers. An experimental (surface potential) and theoretical (Gouy-Chapman) approach.

Ionization of the acidic phospholipid phosphatidylglycerol has been studied by measuring the surface potential of monomolecular films of the lipid as a function of the aqueous subphase pH and the concentration of monovalent cations (Li, Na, Cs). It is shown that the experimental data can be interpreted by means of the Gouy-Chapman theory in its simplest formulation, provided an adsorption of cations at the membrane surface is accounted for. This allows us to predict the ionization state of the lipid for given ionic conditions in the subphase. Above pH 4, for subphase ion concentration higher than 10 mM, or for ion concentrations above 0.1 mM at pH 5.6, phosphatidylglycerol is fully deprotonated. Within the limits of our theoretical approach, association constants of the cations to the lipid lie around 0.1-0.6 M-1.

The use of fluorescence anisotropy decay of poly d(A-T) ethidium bromide complex to estimate the unwinding angle of the double helix.

We measured the fluorescence decay under polarized light, of ethidium bromide bound to the poly d(A-T) isolated from Cancer Pagurus. The decay of the whole fluorescence is a single exponential function revealing a good homogeneity of the binding sites. The anisotropy decay due to energy transfers between the ethidium bromide molecules bound to a same poly d(A-T) molecule has been analysed, with a Monte Carlo calculation. We found the dye unwinds the poly d(A-T) duplex by an angle of 17 degrees plus or minus 2 degrees. This result is in agreement with the value previously found in the case of calf thymus DNA-ethidium bromide complex, although the base compositions of the two nucleic acids are different.