Competition between erythromycin and virginiamycin for in vitro binding to the large ribosomal subunit

When the S component of virginiamycin binds in vitro to the 50 S ribosomal subunit, a change of fluorescence intensity proportional to the amount of complex formed occurs. Erythromycin competes with virginiamycin S for attachment to ribosomes, and removes previously bound virginiamycin S from its target, as revealed by spectrofluorimetric analysis. The 50 S subunits which are incubated with the M component of virginiamycin (50 S*) have an increased affinity for virginiamycin S (the association constants of virginiamycin S with ribosomes are 2.5 x 10(6) M-1 in the absence of virginiamycin M, and 15 x 10(6) M-1 in its presence). Erythromycin does not compete with virginiamycin S for attachment to 50 S* subunits nor is it able to remove virginiamycin S previously bound to the 50 S* subunit. Thus, virginiamycin M produces a change in ribosomes, which results in a tighter complex virginiamycin S-50 S* subunit. Such change does not require the presence of virginiamycin M, however, as shown by the observation that ribosomes to which labeled virginiamycin M is transiently linked bind virginiamycin S in a form that cannot be removed by erythromycin.     

Determination of the distribution of protein and nucleic acid in the 70 S ribosomes of Escherichia coli and their 30 S subunits by neutron scattering.

Neutron small-angle scattering of the 70 S Escherichia coli ribosomes and of its smaller 30 S subunit has been measured in $\text{H}{}_2\text{O}{}^2\text{H}{}_2\text{O}$ mixtures. A linear dependence of the square of the radius of gyration on the reciprocal of the contrast is found, which is qualitatively similar to the results from contrast variation with the larger 50 S subunit. The slope α in this plot is a measure of radial segregation of RNA and proteins. It is most pronounced with the 50 S subunit. The 30 S particle appears to be more homogeneous, whereas the 70 S ribosome assumes an intermediate value of α. Neither the 30 S and 50 S subunits nor the 70 S ribosome show a significant separation of the centres of mass of their RNA part and proteins. A quantitative comparison of the parameters obtained suggest that the interaction between the two subunits and the 70 S ribosomes does not involve any major change in the latter.     

A spectrofluorimetric study of the interaction between virginiamycin S and bacterial ribosomes

Summary Virginiamycin S (VS, a type B component of the synergistin group of antibiotics) is fluorescent in solution: the fluorescence intensity is proportional to VS concentration. The intensity of VS fluorescence was found to increase upon addition of 50S ribosomal subunits, and this variation (I₄₁₆nm) to be proportional to the concentration of 50S subunits. This new technique was, then, used to measure the binding reaction of VS to ribosomes. Similar patterns of link age were obtained for ribosomes and large subunits, whereas very little fixation to 30S particles was detected. The binding reaction was virtually instantaneous at any temperature, and, for saturating VS, was not influenced by Mg⁺⁺ concentration in the range 1 to 20 mM, nor by the replacement of 100 mM K⁺ with NH ₄ ⁺ . The association constant of VS to 50S particles was found to be KA=2.5 × 10⁶M^–1, and from the Scatchard plot a value of 0.9 was calculated, which points to a stoichiometric reaction leading to 1 mole VS bound per mole of 50S particles. upon fixation of virginiamycin M (VM, a type A component of the synergistin group of antibiotics), the I of the VS-ribosome complex was increased, and a KA =15 × 10⁶M^–1 was recorded for the association constant of VS to 50S particles. Such sixfold increase in the affinity of ribosomes for VS may account for the synergistic effect of the 2 virginiamycin components in sensitive bacteria.     

Mitochondrial evidence for distinct phylogeographic units in the endangered Malagasy poison frog Mantella bernhardi

Mantella bernhardi is an endemic species of Malagasy poison frog threatened by loss and fragmentation of its natural habitat and collection for the pet trade. It is classified as threatened according to the International Union for Conservation of Nature and Natural Resources (IUCN) categories and included in Appendix II of the Convention on the International Trade of Endangered Species (CITES). A recent survey has increased the known distributional range of the species from one to eight populations across southeastern Madagascar, but little is known about its biology and genetic diversity. Here we estimate inter- and intrapopulation mitochondrial genetic variation of four populations. Populations from the northern and southern parts of the distributional range showed a high degree of divergence (maximum of 11.35% in cytochrome b) and were recovered as reciprocally monophyletic groups. Nine haplotypes were detected in the northern and 12 in the southern populations. The population from Ranomafana National Park showed the lowest number of haplotypes and nucleotide diversity, and shared its most common haplotype with the second northern population from Tolongoina. All the other detected haplotypes were unique to each of the four populations. This suggests the existence of important barriers to gene flow, pre-dating human colonization of Madagascar at about 2000 years ago, in distinct contrast to other Mantella species that show a high degree of haplotype sharing throughout their range. The continued habitat fragmentation within the distribution range of M. bernhardi prevents any connection between its populations. Our data indicate the existence of at least two different management units for conservation in this species, corresponding to the North and South of its distribution range, and highlight the existence of strong regional endemism in southeastern Madagascar.     

Classification of prostate magnetic resonance spectra using Support Vector Machine

Prostate cancer is the most common cancer in men over 50 years of age and it has been shown that nuclear magnetic resonance spectra are sensitive enough to distinguish normal and cancer tissues. In this paper, we propose a classification technique of spectra from magnetic resonance spectroscopy. We studied automatic classification with and without quantification of metabolite signals. The dataset is composed of 22 patient datasets with a biopsy-proven cancer, from which we extracted 2464 spectra from the whole prostate and of which 1062 were localised in the peripheral zone. The spectra were manually classed into 3 different categories by a spectroscopist with 4 years experience in clinical spectroscopy of prostate cancer: undetermined, healthy and pathologic. We used different preprocessing methods (module, phase correction only, phase correction and baseline correction) as input for Support Vector Machine and for Multilayer Perceptron, and we compared the results with those from the expert. If we class only healthy and pathologic spectra we reach a total error rate of 4.51%. However, if we class all spectra (undetermined, healthy and pathologic) the total error rate rises to 11.49%. We have shown in this paper that the best results are obtained using the pre-processed spectra without quantification as input for the classifiers and we confirm that Support Vector Machine are more efficient than Multilayer Perceptron in processing high dimensional data.     

Synthesis and properties of a new cleavable nucleic acid-protein crosslinking reagent

A new compound, dithiobis[9-(2-ethylenecarbamoylethylamino)-2,3-dimethoxy-6-azido-acridine], was synthesized and used in some preliminary experiments to form cleavable complexes between nucleic acids and proteins. In a first step the azidoacridine moiety of the reagent intercalates between the bases of nucleic acids and is then bound by reaction of the azido group. The disulfide group of the reagent is simultaneously converted under reducing conditions into a thiol which, in a second step, can be bound by oxidation to -SH groups of a vicinal protein (additional -SH groups can be inserted in the protein using 2-iminothiolane). The nucleic acid-protein complexes thus formed can be redissociated by reduction. The potential applications of this new cleavable crosslinking reagent could be extended to topographical investigations of any biological structure composed of nucleic acids and proteins.