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.

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.

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.

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.

Retinoic acid: enhancement of a tumor and inhibition of interferon's antitumor action.

The effect of trans-retinoic acid on the growth of P388 lymphoid tumors in inbred female DBA/2 mice in the presence or absence of interferon (FN) treatment was studied. This acid derivative of vitamin A enhanced local tumor growth. trans-retinoic acid also partially reversed IFN protection against tumor growth and mortality.

Control of Polyomavirus T-antigen and DNA synthesis in mouse embryo fibroblast cells by vitamin A.

Vitamin A (retinoic acid, 10(-6) M) treatment of confluent mouse embryo cells for only 7 h resulted in optimal inhibition of Polyomavirus replication. Depending upon the input multiplicity of virus, one could wait until between 12 and 18 h postinfection to add vitamin A and still observe maximal inhibition of virus yields. Taken together, and assuming the same kinetics before and after virus infection, these results suggested that the inhibitory action of vitamin A occurred between 19 and 25 h into the Polyomavirus replication cycle. In this model system, such a time corresponded to the onset of T-antigen expression and virus-induced cellular DNA synthesis. Analysis of both viral and virus-induced cellular DNA synthesis by the method of Hirt (J. Mol. Biol., 26: 365-369, 1967) and by cesium chloride gradients suggested that vitamin A preferentially inhibited viral, more than virus-induced cellular, DNA synthesis in confluent cell monolayers. Vitamin A also concomitantly inhibited Polyomavirus T-antigen expression in such confluent cultures. In contrast, viral DNA synthesis and infectious virus yields were not significantly inhibited by vitamin A in subconfluent cell cultures. The antagonistic effect of vitamin A on Polyomavirus replication in confluent monolayers could be blocked with cycloheximide, a reversible protein synthesis inhibitor. This suggested that vitamin A inhibition of Polyomavirus replication was indirect and mediated by a newly synthesized protein. Taken together, these results suggest that vitamin A induced a protein in confluent, but not subconfluent, cells, which blocked the expression of Polyomavirus T-antigen. Decreased amounts of T-antigen most likely reduced Polyomavirus and cellular DNA synthesis and virus yield.

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.

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.

Ehlers-Danlos syndrome during pregnancy: a case report and review of the literature.

Since Ehlers-Danlos syndrome encompasses a wide spectrum of disease and only has an estimated incidence of approximately 1:150,000 (Beighton, 1968a), there is a paucity of data from large studies on which to base firm conclusions about the risks of pregnancy in association with the syndrome. Most reports refer to women with the gravis (type I) and ecchymotic (type IV) forms, unlike pregnancy in association with the mitis (type II), which is rarely reported despite the fact that it constitutes 35 per cent of all cases of Ehlers-Danlos syndrome (Beighton, 1970). We suspect this is because complications with the mitis form are rare. Women with the two severe forms of the syndrome, mentioned above, risk hemorrhage, severe trauma at delivery, and preterm labor, but whether this is the case in the mitis form is unproved. Our patient's course was uneventful, delivery by cesarean section being undertaken because of her hip fixity. Tyson (1972) suggested that delivery by cesarean section was indicated in all cases of Ehlers-Danlos syndrome, but this pertains to the severe forms of the syndrome. The question of vaginal delivery in the mitis form remains unanswered. We would hope that in future reports of pregnancy in association with Ehlers-Danlos syndrome that patients are classified according to type so that prognosis, particularly in the mitis form, can be more accurately assessed.

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.

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.

Family health and family size.

PIP: There is a greater risk to both mother and child as parity increases, especially after the fifth pregnancy. Hazards of high parity include placenta praevia, hemorrhage, prolasped cord, abnormal presentation of the fetus, uterine rupture, and postpartum inertia with heavy bleeding. Although grand multiparas need special obstetrical care, they are often reluctant to deliver in the hospital, and are ususally from the poorer classes where other health and socioeconomic problems complicate the problem. Because of the resulting problems associated with high parity, and the subsequent health problems to the offspring, there is a great need to achieve universal acceptance of the desirability of planning and controlling family size.^ieng