Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.     

Mammalian lignans: possible involvement in endogenous digitalis activity

Lignans are natural products, some of which were recently discovered in animal urines, semen and blood plasma. We investigated the actions of animal lignans obtained by total synthesis or extracted from urines of pregnant women on Na+, K+-ATPase in human red cells and human and guinea-pig heart cell membranes. Some of the tested lignans (enterolactone, prestegane B and 3-O-methyl enterolactone) inhibited Na+, K+-pump activity in human red cells with IC50 ranging from 5 to 9 X 10(-4) M. The IC50 for ouabain (7 X 10(-7) M) was not modified by addition of lignans. Enterolactone inhibited Na+, K+-ATPase activity in human and guinea pig heart membranes. It also displaced [3H]-ouabain binding from human heart with IC50 = 1.5 X 10(-4) M. The apparent dissociation rate constants (kd) of [3H]-ouabain were not different in presence of digoxin or enterolactone. Enterolactone exhibited a poor cross reactivity against antidigoxin antibodies. The aglycones of the lignans studied here were slight inhibitors of the Na+, K+-ATPase. However, we cannot exclude that a glycosyl- (and/or butenolide-) derivative of enterolactone could be one "endogenous ouabain-like" factor.     

A new spatial structure for the axial methionine observed in cytochrome c5 from Pseudomonas mendocina. Correlations with the electronic structure of heme c

Cytochrome c5 from Pseudomonas mendocina has been isolated and the coordination geometry at the heme iron was investigated by 1H nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with that of other c-type cytochromes. In contrast, a new structure was observed for the axial methionine. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa, which also contain S-chiral methionine, the spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine is markedly different. Analysis of the electron spin density distribution in ferricytochrome c5 in the light of this new coordination geometry provides additional support for the hypothesis that the electronic structure of heme c is primarily governed by the orientation of the sp3 lone-pair orbital of the axial sulfur atom with respect to the heme plane.     

Consequences of herbivory in the mountain birch (Betula pubescens ssp tortuosa): importance of the functional organization of the tree

Summary Three types of experiments indicate that the functional organization of the mountain birch may influence the ways in which the tree responds to simulated or natural herbivory. The first experiment showed that herbivory to both short and long shoot leaves affects plant development but, because growth largely proceeds by resources of the previous year, is manifested only in the year following the damage. The second experiment showed that even partial damage to a single long shoot leaf caused the axillary bud of that leaf to produce a shorter shoot the next year. Therefore, the value of a leaf depends also on the organ which it is subtending. In the third experiment we manipulated the apical dominance of shoots in ramets and caused improvement to leaf quality in extant shoots. Ramets within a tree responded individually, probably mediated by disturbance of the hormonal control because removal of apical buds elicited the response although removal of the same number of basal buds did not. Induced amelioration is a different response to induced resistance. The two responses are triggered by different cues and may occur in the same plant. By altering hormonal balance of shoots it is potentially possible for herbivores to induce amelioration of food quality. The ways in which herbivory is simulated may explain variability of results obtained when herbivory-induced responses in plants have been studied.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Strange limits of stability in host-parasitoid systems

The classical Nicholson-Bailey model for a two species host-parasitoid system with discrete generations assumes random distributions of both hosts and parasitoids, randomly searching parasitoids, and random encounters between the individuals of the two species. Although unstable, this model induced many investigations into more complex host-parasitoid systems. Local linearized stability analysis shows that equilibria of host parasitoid systems within the framework of a generalized Nicholson-Bailey model are generally unstable. Stability is only possible if host fertility does not exceede ⁴=54.5982 and if superparasitism is unsuccessful. This special situation has already been discovered by Hassell et al. (1983) in their study of the effects of variable sex ratios on host parasitoid dynamics. We discuss global behaviour of the Hassell-Waage-May model using KAM-theory and illustrate its sensitivity to small perturbations, which can give rise to radically different patterns of the population dynamics of interacting hosts and parasitoids.