Ash production by attrition in volcanic conduits and plumes.

Tephra deposits result from explosive volcanic eruption and serve as indirect probes into fragmentation processes operating in subsurface volcanic conduits. Primary magmatic fragmentation creates a population of pyroclasts through volatile-driven decompression during conduit ascent. In this study, we explore the role that secondary fragmentation, specifically attrition, has in transforming primary pyroclasts upon transport in volcanic conduits and plumes. We utilize total grain size distributions from a suite of natural and experimentally produced tephra to show that attrition is likely to occur in all explosive volcanic eruptions. Our experimental results indicate that fine ash production and surface area generation is fast (<15 min) thereby rapidly raising the fractal dimension of tephra deposits. Furthermore, a new metric, the Entropy of Information, is introduced to quantify the degree of attrition (secondary fragmentation) from grain size data. Attrition elevates fine ash production which, in turn, has consequences for eruption column stability, tephra dispersal, aggregation, volcanic lightening generation, and has concomitant effects on aviation safety and Earth's climate.

A non-natural variant of human lysozyme (I59T) mimics the in vitro behaviour of the I56T variant that is responsible for a form of familial amyloidosis.

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains. Although more stable in its native structure than the naturally occurring variants that give rise to a familial form of systemic amyloidosis, I59T possesses many attributes that are similar to these disease-associated species. In particular, under physiologically relevant conditions, I59T populates transiently an intermediate in which a region of the structure unfolds cooperatively; this loss of global cooperativity has been suggested to be a critical feature underlying the amyloidogenic nature of the disease-associated lysozyme variants. In the present study, we have utilised this variant to provide direct evidence for the generic nature of the conformational transition that precedes the ready formation of the fibrils responsible for lysozyme-associated amyloid disease. This non-natural variant can be expressed at higher levels than the natural amyloidogenic variants, enabling, for example, singly isotopically labelled protein to be generated much more easily for detailed structural studies by multidimensional NMR spectroscopy. Moreover, we demonstrate that the I59T variant can readily form fibrils in vitro, similar in nature to those of the amyloidogenic I56T variant, under significantly milder conditions than are needed for the wild-type protein.

Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris.

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.

Crystallographic refinement by knowledge-based exploration of complex energy landscapes.

Although X-ray crystallography remains the most versatile method to determine the three-dimensional atomic structure of proteins and much progress has been made in model building and refinement techniques, it remains a challenge to elucidate accurately the structure of proteins in medium-resolution crystals. This is largely due to the difficulty of exploring an immense conformational space to identify the set of conformers that collectively best fits the experimental diffraction pattern. We show here that combining knowledge-based conformational sampling in RAPPER with molecular dynamics/simulated annealing (MD/SA) vastly improves the quality and power of refinement compared to MD/SA alone. The utility of this approach is highlighted by the automated determination of a lysozyme mutant from a molecular replacement solution that is in congruence with a model prepared independently by crystallographers. Finally, we discuss the implications of this work on structure determination in particular and conformational sampling and energy minimization in general.

Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme.

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.

Optimal Response to Cardiac Arrest study: defibrillation waveform effects.

INTRODUCTION: Advances in early defibrillation access, key to the "Chain of Survival", will depend on innovations in defibrillation waveforms, because of their impact on device size and weight. This study compared standard monophasic waveform automatic external defibrillators (AEDs) to an innovative biphasic waveform AED. MATERIAL AND METHODS: Impedance-compensated biphasic truncated exponential (ICBTE) and either monophasic truncated exponential (MTE) or monophasic damped sine (MDS) AEDs were prospectively, randomly assigned by date in four emergency medical services. The study design compared ICBTE with MTE and MDS combined. This subset analysis distinguishes between the two classes of monophasic waveform, MTE and MDS, and compares their performance to each other and to the biphasic waveform, contingent on significant overall effects (ICBTE vs. MTE vs. MDS). Primary endpoint: Defibrillation efficacy with < or =3 shocks. Secondary endpoints: shock efficacy with < or =1 shock, < or =2 shocks, and survival to hospital admission and discharge. Observations included return of spontaneous circulation (ROSC), refibrillation, and time to first shock and to first successful shock. RESULTS: Of 338 out-of-hospital cardiac arrests, 115 had a cardiac aetiology, presented with ventricular fibrillation, and were shocked by an AED. Defibrillation efficacy for the first "stack" of up to 3 shocks, for up to 2 shocks and for the first shock alone was superior for the ICBTE waveform than for either the MTE or the MDS waveform, while there was no difference between the efficacy of MTE and MDS. Time from the beginning of analysis by the AED to the first shock and to the first successful shock was also superior for the ICBTE devices compared to either the MTE or the MDS devices, while again there was no difference between the MTE and MDS devices. More ICBTE patients achieved ROSC pre-hospital than did MTE patients. While the rates of ROSC were identical for MTE and MDS patients, the difference between ICBTE and MDS was not significant. Rates of refibrillation and survival to hospital admission and discharge did not differ among the three populations. CONCLUSIONS: ICBTE was superior to MTE and MDS in defibrillation efficacy and speed and to MTE in ROSC. MTE and MDS did not differ in efficacy. There were no differences among the waveforms in refibrillation or survival.

Multicenter, randomized, controlled trial of 150-J biphasic shocks compared with 200- to 360-J monophasic shocks in the resuscitation of out-of-hospital cardiac arrest victims. Optimized Response to Cardiac Arrest (ORCA) Investigators.

BACKGROUND: In the present study, we compared an automatic external defibrillator (AED) that delivers 150-J biphasic shocks with traditional high-energy (200- to 360-J) monophasic AEDs. METHODS AND RESULTS: AEDs were prospectively randomized according to defibrillation waveform on a daily basis in 4 emergency medical services systems. Defibrillation efficacy, survival to hospital admission and discharge, return of spontaneous circulation, and neurological status at discharge (cerebral performance category) were compared. Of 338 patients with out-of-hospital cardiac arrest, 115 had a cardiac etiology, presented with ventricular fibrillation, and were shocked with an AED. The time from the emergency call to the first shock was 8.9+/-3.0 (mean+/-SD) minutes. CONCLUSIONS: The 150-J biphasic waveform defibrillated at higher rates, resulting in more patients who achieved a return of spontaneous circulation. Although survival rates to hospital admission and discharge did not differ, discharged patients who had been resuscitated with biphasic shocks were more likely to have good cerebral performance.

The solution structure of a class II major histocompatibility complex superantigen binding domain.

We have used 600 MHz 1H NMR spectroscopy data to determine the solution structure of a 31-residue domain of a murine class II major histocompatibility (MHC) protein. This domain, I-Ab(beta)-(60-90), binds to the superantigen staphylococcal enterotoxin A. Distance geometry and dynamical simulated annealing calculations were performed using NOESY- and COSY-deduced constraints. I-Ab(beta)-(60-90), which is mostly alpha-helical, is more similar to the corresponding region of the class II MHC protein HLA-DR1 than to the class I MHC protein HLA-A2. Arg-72 and Arg-80 lie on the same side of the helix and face away from the antigenic peptide binding groove. His-81, implicated in both superantigen and peptide binding, is located midway between the surface defined by Arg-72/Arg-80 and residues that define the inside of the peptide binding groove, allowing for its participation in both types of binding.

Bodywork--the art of touch.

Massage, neuromuscular therapy, Trager, and Bowen work reduce stress, relieve pain, rebalance the body, and restore a sense of general well-being. They can be used by themselves to promote physical and emotional health or with conventional medical care to restore health. This article examines the differences and similarities among these four therapies and concludes with case histories to illustrate the use of bodywork in primary care.

Identification of binding domains on the superantigen, toxic shock syndrome-1, for class II MHC molecules.

Toxic shock syndrome toxin-1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. In order to determine the regions on the TSST-1 molecule involved in binding to class II MHC, seven overlapping peptides of the entire TSST-1 molecule were synthesized and tested for their ability to compete with 125I-TSST-1 for binding to class II MHC on murine A20 cells and HLA on Raji cells. Peptides corresponding to N-terminal amino acid residues 39 through 68 and C-terminal residues 155 through 194 competed with 125I-TSST-1 for binding to class II MHC. Also, binding studies with class II MHC beta-chain peptides indicate that regions encompassed by I-A beta b(30-60) and I-A beta b(60-90) are binding regions for TSST-1. Thus, we have identified binding domains on the TSST-1 molecule for class II MHC molecule receptors on antigen presenting cells.

Evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A.

Circular dichroism (CD) spectra of class II MHC peptides revealed the alpha-helical conformation of superantigen-binding peptides I-A beta b(60-90), I-A beta b(65-85), and I-A alpha b(51-80), but not the nonbinding peptide I-A beta b(80-100). These CD spectra provide biophysical evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A (SEA). Alanine-substituted analogs of the SEA binding-site peptide, I-A beta b(65-85), were used to implicate beta-chain residues 72 and 80 in class II MHC-SEA binding. The data support SEA binding away from the class II antigen binding cleft along the faces of the alpha-helices.

Staphylococcal enterotoxin superantigens.

Staphylococcal enterotoxins (SE) are a family of structurally related proteins that are produced by Staphylococcus aureus. They play a role in the pathogenesis of food poisoning and are the most potent activators of T lymphocytes known. The receptors for SE on antigen-presenting cells are major histocompatibility complex class II molecules. Recent studies have shown that a complex of SE and major histocompatibility complex class II molecules is required for binding to the variable region of the T cell antigen receptor beta-chain. SE mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of SE toxicity. The minor lymphocyte-stimulating "endogenous" self-superantigen has recently been shown to be a retroviral gene product, so that this too is apparently a microbial superantigen. An understanding of the mechanism of action of these microbial superantigens has implications for normal and pathological immune functions.

Staphylococcal enterotoxin microbial superantigens.

Staphylococcal enterotoxins are a family of structurally related proteins that are produced by Staphylococcus aureus. In addition to their role in the pathogenicity of food poisoning, these microbial superantigens have profound effects on the immune system, which makes them useful tools for understanding its mechanism of action. These molecules (24-30 kDa) are highly hydrophilic and exhibit low alpha helix and high beta pleated sheet content, suggesting a flexible, accessible structure. Staphylococcal enterotoxins are among the most potent activators of T lymphocytes known. The receptors for staphylococcal enterotoxins on antigen-presenting cells are major histocompatibility complex (MHC) class II molecules. Further, the alpha-helical regions of the class II molecule are essential for function and appear to interact directly with the NH2-terminal region of staphylococcal enterotoxins such as SEA. Recent studies have shown that a complex of staphylococcal enterotoxin and MHC class II molecules is required for binding to the V beta region of the T cell antigen receptor. Staphylococcal enterotoxin mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of toxicity. The mouse minor lymphocyte stimulating (M1s) "endogenous" self-superantigen has been shown to be a retroviral gene product, so this too is apparently a microbial superantigen. An understanding of the mechanisms of action of these microbial superantigens has implications for normal and pathological immune functions.

Both alpha-helices along the major histocompatibility complex binding cleft are required for staphylococcal enterotoxin A function.

The superantigen staphylococcal enterotoxin A (SEA) requires interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously used the synthetic peptide approach to establish one side of the hypothetical class II foreign-antigen binding cleft, alpha-helical region 65-85 of the beta chain, as a binding site involved in accessory cell presentation of SEA to T cells. To further characterize the structural basis for MHC-SEA interaction we have examined the role of the alpha-helical regions of the class II alpha and beta chains in SEA function. Using the synthetic peptide approach, we have found that both alpha-helical regions are required for SEA-induced proliferation. Their corresponding peptides directly bound SEA. Although the beta-chain peptides were able to inhibit SEA binding to human and mouse cells, the alpha-chain peptides were not. The data suggest that the alpha-helices along both sides of the hypothetical class II MHC molecule binding cleft are required for SEA-induced function, whereas the beta-chain alpha-helix is sufficient for SEA binding. A model of superantigen presentation is proposed wherein the MHC beta chain, possibly region 70-80, interacts with SEA region 1-45, whereas another region of SEA binds region 51-80 of the alpha chain.

Structural basis for differential binding of staphylococcal enterotoxin A and toxic shock syndrome toxin 1 to class II major histocompatibility molecules.

The related staphylococcal toxins staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) are microbial superantigens. They require interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously identified a binding site on SEA, the N-terminal 45 amino acids, as well as its corresponding receptor on the MHC antigen, residues 65-85 of the beta chain. To further characterize the structural basis for SEA binding to class II MHC molecules we have examined its relationship to TSST-1 binding. Both toxins bound similarly to murine A20 cells, but blockage of binding was observed only with the homologous toxin, which suggests that the binding sites for the two toxins on A20 cells are distinct. In contrast, specific binding of SEA was greater than that of TSST-1 on human Raji cells. Further, SEA was a better inhibitor of TSST-1 binding than was TSST-1 itself at low concentrations, but TSST-1 only minimally inhibited SEA binding. The data suggest that TSST-1 interacts with Raji cells at an SEA binding site, but with a lower affinity. The peptides SEA-(1-45) and I-A beta b-(65-85) were capable of blocking SEA binding on both A20 and Raji cells, but blockage was more effective on A20 cells. Neither peptide was capable of blocking TSST-1 binding on either cell line. The data are compatible with a model in which SEA has a binding site on A20 cells involving SEA-(1-45) and I-A beta b-(65-85) which is distinct from that which binds TSST-1, while at least two binding sites are present on Raji cells. One site involves predominantly the residue 1-45 region on SEA and the 65-85 region of the MHC beta chain, while the other site involves both a different region on the SEA molecule and a different site on the class II MHC molecule to which it binds. This latter site also binds TSST-1.

The I-A beta b region (65-85) is a binding site for the superantigen, staphylococcal enterotoxin A.

Ia antigen is a receptor for the superantigen staphylococcal enterotoxin A (SEA). Peptides I-A beta b(30-60), I-A beta b(50-70), I-A beta b(65-85), and I-A beta b(80-100) of the MHC class II antigen beta chain on mouse (H-2b) accessory cells were synthesized. Only I-A beta b(65-85) inhibited SEA binding to the mouse B-cell lymphoma line, A20 (H-2d) and the human Burkitt's lymphoma line, Raji (HLA-DR). The I-A beta b(65-85) sequence is a predicted alpha-helix along the hypothetical antigen binding cleft of the Ia molecule. I-A beta b(65-85) also directly and specifically bound both the intact SEA molecule and its Ia binding site, represented by the peptide SEA(1-45). The results suggest that I-A beta b region (65-85) is a necessary site for Ia molecular interaction with the superantigen SEA. Further, the data suggest that the same helical region of other Ia antigens binds SEA irrespective of haplotype and species.

Site of nonrestrictive binding of SEA to class II MHC antigens.

We have used the synthetic peptide approach to show that the N-terminal 45-amino acids of staphylococcal enterotoxin A (SEA), SEA(1-45), constitute an important part of its binding site on class II major histocompatibility complex (MHC) molecules. SEA(1-45) and to a lesser extent SEA(1-27) were able to displace SEA from HLA-DR on Raji cells as assessed by flow cytometry and to compete with radiolabeled SEA for interaction with HLA-DR in a direct binding assay. Specific binding of SEA to Ia on murine A-20 cells could be inhibited by the same peptides [i.e. SEA(1-45) greater than SEA(1-27)] that blocked binding to HLA-DR. Therefore, different class II MHC molecules associate with the same functional site on SEA. Further, an ELISA system was used to demonstrate that SEA(1-45) is able to directly bind to a mouse synthetic I-A beta b peptide, I-A beta b (65-85), which contains a binding site of the class II MHC molecule involved in SEA presentation to T cells. Thus, we have localized a site on SEA that is involved in selective surface association with class II MHC antigens and identified the region on the class II MHC antigen to which that site binds.

Localization of an immune functional site on staphylococcal enterotoxin A using the synthetic peptide approach.

Using the synthetic peptide approach, we have identified a part of the staphylococcal enterotoxin A (SEA) molecule that is responsible for stimulation of T cell proliferation and induction of the lymphokine IFN-gamma. Peptides were synthesized corresponding to amino acids 1 to 27, SEA(1-27), and 28 to 45, SEA(28-45). Both peptides were tested for direct competition with SEA for blockage of SEA induced proliferation and production of IFN-gamma by T cells. Further, antibodies were produced to the peptides and tested for their ability to bind to SEA and block SEA function. SEA (1-27), but not SEA (28-45), blocked proliferation of human peripheral T cells and induction of IFN-gamma by the T cell line, L12-R4. The inhibitory effects were specific, because SEA (1-27) did not inhibit the induction of T cell proliferation by the mitogen PHA. Consistent with the direct inhibition of function, antibodies to SEA (1-27), but not SEA (28-45), neutralized the mitogenic activity of SEA on human PBL. The data suggest that a functional site on SEA that is responsible for its modulation of T cell function involves the N-terminal 27 amino acids. Residues 1 to 27 of SEA could potentially interact at either the level of the TCR or may block the proposed binding of SEA to class II MHC Ag, based on recent data showing that these molecules are involved in SEA-induced proliferation.

Causal association of interferon-gamma with tumor regression.

Mouse interferon-gamma (MuIFN-gamma) can cause the rejection of malignant cells in vivo. The evidence presented here in support of this claim includes, first, that spontaneous regression of MSC sarcomas was associated with high intratumoral concentrations of endogenously-produced MuIFN-gamma. By contrast, progressively growing, lethal neoplasms of the same kind invariably contained very little IFN-gamma. Second, spontaneously regressing MSC sarcomas were converted into progressively growing, lethal neoplasms by injecting mice with a monoclonal antibody that neutralized the biological effects of endogenous IFN-gamma. Another monoclonal antibody that bound to, but did not neutralize, mouse IFN-gamma had no effect on the course of tumor regression. Together, these data causally relate MuIFN-gamma to the successful rejection of malignant cells in vivo. They also suggest that findings of poor therapeutic efficacy for IFN-gamma are probably attributable to problems other than an intrinsic lack in the biological activity of the lymphokine.