CD and (13)C(alpha)-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of alpha-tropomyosin

Synthesis and CD and (13)C(alpha)-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide ((190)Tm(254)) comprising residues 190-254 of the alpha-tropomyosin chain. CD on a version disulfide cross-linked via the N-terminal cysteine side chains indicates that this dimer is highly helical and melts near 48 degrees C. The CD is independent of peptide concentration, showing that association of (190)Tm(254) stops at the two-strand level. Similar studies on the reduced version show much lower helix content at low temperature, melting points below room temperature, and the expected concentration dependence. The observed melting temperature of the reduced peptide is far below (by 27 degrees C) that expected from an extant analysis of calorimetry data on parent tropomyosin that designates (190)Tm(254) as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for (190)Tm(254) are combined with extant data for other subsequences argue decisively against the concept of specific independently melting blocks within the tropomyosin chain. The data for (190)Tm(254) also serve to test recent ideas on the sequence determinants of structure and stability in coiled coils. Analysis shows that some ideas, such as the stabilizing effect of leucine in the d heptad position, find support, but others--such as the destabilizing effect of alanine in d, the dimer-disfavoring effect of beta-branching in d and its dimer-favoring effect in a, and the dimer-directing effect of asparagine in a--are more questionable in tropomyosin than in the leucine zipper coiled coils. (13)C(alpha)-NMR data at two labeled sites, L228(d) and V246(a), of (190)Tm(254) display well-separated resonances for folded and unfolded forms at each site, indicating that the transition is slow on the NMR time scale and thus demonstrating the possibility of obtaining thermodynamic and kinetic information on the transition at the residue level.     

Specificity of gene regulation

The physiologically coordinated expression of our genome requires exquisite regulation of gene specificity. Recent advances demonstrate that this formidable task is accomplished by diverse mechanisms and networks that operate at distinct levels within the nucleus.     

Reducing malaria by mosquito-proofing houses

Sometimes, valuable lessons from history are forgotten, remain unknown, or worse, are ignored. This article reminds us of the pioneering work of Angelo Celli at the end of the 19th century, who demonstrated that people could be protected from malaria by screening their homes against mosquitoes. Since then, public health scientists have continued to show that simple changes in house design have the potential for protecting people against this life-threatening disease. Yet today, this type of intervention remains virtually ignored. The literature reviewed here demonstrates the enormous potential of these methods to reduce malaria, in the hope that it will stimulate scientific debate and further research.     

Ash content modulation of torsionally derived effective material properties in cortical mouse bone

The purpose of this study was to evaluate the effects of isolated alterations in mineral content on mouse bone torsional properties. The femora and tibiae from 25 eight-week-old male A/J strain mice were divided into five groups and selectively decalcified from 5% to 20%. The right femora were then tested to failure in torsion while the tibiae were ashed to determine final mineral content of the decalcified bones. Contralateral femora were serially cross-sectioned to determine geometric properties, and effective material properties were then calculated from the geometric and structural properties of each femoral pair. We found that the relationship between ash content and effective shear modulus or maximum effective shear stress could best be characterized through a power law, with an exponential factor of 6.79 (R2 = 0.85) and 4.04 (R2 = 0.67), respectively. This indicates that in a murine model, as with other species, small changes in ash content significantly influence effective material properties. Furthermore, it appears that (in adolescent A/J strain mice) effective shear modulus is more heavily affected by changes in mineralization than is maximum effective shear stress when these properties are derived from whole bone torsional tests to failure.     

α-Helix-to-random-coil transitions of two-chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross-linked selectively at C-190

A method is described for preparation of a species of β tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomyosin, comprising αα and αβ species, is oxidized with ferricyanide, disulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the β-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (DTT), unfolded in urea, and α and β chains separated by ion-exchange chromatography. (4) The C36-blocked β chains are refolded by dialysis. (5) The refolded, C36-blocked ββ species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, C190-cross-linked ββ product is separated from contaminating species—mostly completely blocked β-chains and multichain cross-linked molecules—by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaDodSO₄/polyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked ββ species and for its DTT-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked ββ species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of C190-cross-linked αα tropomyosin. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) ββ species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. It is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the physics.     

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.     

Effect of the beta-gamma phosphate bond of ATP on synthesis of leader RNA and mRNAs of vesicular stomatitis virus.

RNA products synthesized in vitro by vesicular stomatitis virus with normal nucleotides or imido analogs were compared by polyacrylamide gel electrophoresis. When imido ATP, which has a nonhydrolyzable beta-gamma bond, was substituted for ATP, leader RNA and DI particle product were synthesized, but appreciable mRNA synthesis was not detected.