Regional stress in a noncircular cylinder.

Several mathematical formulas are presented for estimating regional average circumferential stress and shear stress in a thick-wall, noncircular cylinder with a plane of symmetry. The formulas require images of exterior and interior chamber silhouettes plus surface pressures. The formulas are primarily intended for application to the left ventricle in the short axis plane near the base (where the meridional radius of curvature is normally much larger than the circumferential radius of curvature) and to blood vessels. The formulas predict stresses in a variety of chambers to within 3% of finite element values determined from a large-scale structural analysis computer program called ANSYS.     

Effect of insulin on human skeletal muscle protein synthesis is modulated by insulin-induced changes in muscle blood flow and amino acid availability

Insulin promotes muscle anabolism, but it is still unclear whether it stimulates muscle protein synthesis in humans. We hypothesized that insulin can increase muscle protein synthesis only if it increases muscle amino acid availability. We measured muscle protein and amino acid metabolism using stable-isotope methodologies in 19 young healthy subjects at baseline and during insulin infusion in one leg at low (LD, 0.05), intermediate (ID, 0.15), or high (HD, 0.30 mUxmin(-1)x100 ml(-1)) doses. Insulin was infused locally to induce muscle hyperinsulinemia within the physiological range while minimizing the systemic effects. Protein and amino acid kinetics across the leg were assessed using stable isotopes and muscle biopsies. The LD did not affect phenylalanine delivery to the muscle (-9 +/- 18% change over baseline), muscle protein synthesis (16 +/- 26%), breakdown, or net balance. The ID increased (P < 0.05) phenylalanine delivery (+63 +/- 38%), muscle protein synthesis (+157 +/- 54%), and net protein balance, with no change in breakdown. The HD did not change phenylalanine delivery (+12 +/- 11%) or muscle protein synthesis (+9 +/- 19%), and reduced muscle protein breakdown (-17 +/- 15%), thus improving net muscle protein balance but to a lesser degree than the ID. Changes in muscle protein synthesis were strongly associated with changes in muscle blood flow and phenylalanine delivery and availability. In conclusion, physiological hyperinsulinemia promotes muscle protein synthesis as long as it concomitantly increases muscle blood flow, amino acid delivery and availability.     

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.     

Nonspecific prion protein-nucleic Acid interactions lead to different aggregates and cytotoxic species

A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP-DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP-D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP-nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP-nucleic acid complexes may shed light on the still mysterious pathology of the prion protein.     

Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells

Conversion of the cellular prion protein (PrP(C)) into its altered conformation, PrP(Sc), is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher beta-sheet content and characteristics similar to those of PrP(Sc). RNA molecules can also interact with PrP and potentially modulate PrP(C) to PrP(Sc) conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP(C)-->PrP(Sc) conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.     

Intriguing nucleic-acid-binding features of mammalian prion protein

In transmissible spongiform encephalopathies, the infectious material consists chiefly of a protein, the scrapie prion protein PrP(Sc), that carries no genetic coding material; however, prions are likely to have accomplices that chaperone their activity and promote the conversion of the cellular prion protein PrP(C) into the disease-causing isoform (PrP(Sc)). Recent studies from several laboratories indicate that PrP(C) recognizes many nucleic acids (NAs) with high affinities, and we correlate these findings with a possible pathophysiological role for this interaction. Thus, of the chaperones, NA is the most likely candidate for prions. The participation of NAs in prion propagation opens new avenues for developing new diagnostic tools and therapeutics to target prion diseases, as well as for understanding the function of PrP(C), probably as a NA chaperone.     

Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C

Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.