Analysis of the variation in meat inspection of pigs using variance partitioning.

According to legal regulations, all slaughtered pigs in the European Union are subject to routine meat inspection at the slaughterhouses. The resulting post-mortem findings are valuable indicators that help improve slaughterhouse and farm management and can be used to establish a feedback system regarding animal health. A sufficiently high quality of meat inspection is therefore imperative, which implies that the results of the inspection must not depend on the person carrying out the examination. The objective of the study at hand is the estimation of the amount of variation in these post-mortem findings that can be attributed to the official meat inspectors. In order to reduce the influence of the heterogeneity in the health state of the pigs, the variation due to the farms of origin was considered in the statistical model as well. The analyzed meat inspection data were recorded by 12 official meat inspectors under real working conditions at an Austrian slaughterhouse. Logistic Multilevel Models with cross-classified random effects were applied to 20 post-mortem findings. On the basis of these models, variance partitioning coefficients (VPCs) were used to estimate the amount of variation in the probabilities of these findings due to meat inspector and farm levels. The estimated VPCs suggest that especially meat inspection of blood aspiration, scalding water lungs, skin lesions and hepatitis can be deemed as not sufficiently standardized. Hardly any variation in meat inspection could be identified for other post-mortem findings, such as pericarditis, peritonitis, arthritis and milkspots.

Exact alpha-error determination for two-stage sampling strategies to substantiate freedom from disease.

Sampling strategies to substantiate freedom from disease are important when it comes to the trade of animals and animal products. When considering imperfect tests and finite populations, sample size calculation can, however, be a challenging task. The generalized hypergeometric formula developed by Cameron and Baldock (1998a) offers a framework that can elegantly be extended to multi-stage sampling strategies, which are widely used to account for disease clustering at herd-level. The achieved alpha-error of such surveys, however, typically depends on the realization of the sample and can differ from the pre-calculated value. In this paper, we introduce a new formula to evaluate the exact alpha-error induced by a specific sample. We further give a numerically viable approximation formula and analyze its properties using a data example of Brucella melitensis in the Austrian sheep population.

Dynamics of intramolecular contact formation in polypeptides: distance dependence of quenching rates in a room-temperature glass.

Quenching of the triplet state of tryptophan by cysteine is an important new tool for measuring the rate of forming a specific contact between amino acids in a polypeptide chain. To determine the length scale associated with this contact, tryptophan was embedded in a room-temperature glass containing a high concentration of cysteine. The decay of the triplet population is extended in time, consistent with a rate coefficient that decreases exponentially with distance. Solving the diffusion equation with this distant-dependent rate reproduces the observed bimolecular rates in water and shows that quenching at low viscosities takes place less than or similar to A from van der Waals contact between the tryptophan and cysteine.

Fast kinetics and mechanisms in protein folding.

This review describes how kinetic experiments using techniques with dramatically improved time resolution have contributed to understanding mechanisms in protein folding. Optical triggering with nanosecond laser pulses has made it possible to study the fastest-folding proteins as well as fundamental processes in folding for the first time. These include formation of alpha-helices, beta-sheets, and contacts between residues distant in sequence, as well as overall collapse of the polypeptide chain. Improvements in the time resolution of mixing experiments and the use of dynamic nuclear magnetic resonance methods have also allowed kinetic studies of proteins that fold too fast (greater than approximately 10(3) s-1) to be observed by conventional methods. Simple statistical mechanical models have been extremely useful in interpreting the experimental results. One of the surprises is that models originally developed for explaining the fast kinetics of secondary structure formation in isolated peptides are also successful in calculating folding rates of single domain proteins from their native three-dimensional structure.

Measuring the rate of intramolecular contact formation in polypeptides.

Formation of a specific contact between two residues of a polypeptide chain is an important elementary process in protein folding. Here we describe a method for studying contact formation between tryptophan and cysteine based on measurements of the lifetime of the tryptophan triplet state. With tryptophan at one end of a flexible peptide and cysteine at the other, the triplet decay rate is identical to the rate of quenching by cysteine. We show that this rate is also close to the diffusion-limited rate of contact formation. The length dependence of this end-to-end contact rate was studied in a series of Cys-(Ala-Gly-Gln)(k)-Trp peptides, with k varying from 1 to 6. The rate decreases from approximately 1/(40 ns) for k = 1 to approximately 1/(140 ns) for k = 6, approaching the length dependence expected for a random coil (n(-3/2)) for the longest peptides.

Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering.

We have used synchrotron radiation, together with stopped-flow and continuous-flow mixing techniques to monitor refolding of lysozyme at pH 5.2. From data measured at times which range from 14 ms to two seconds, we can monitor changes in the size, the shape and the pair distribution function of the polypeptide chain during the folding process. Comparison of the results with the properties of native and GdmCl-unfolded lysozyme shows that a major chain collapse occurs in the dead-time of mixing. During this process about 50 % of the change in radius of gyration between the unfolded protein and the native state occurs and the polypeptide chain adopts a globular shape. Time-resolved fluorescence spectra of this collapsed state suggest that the hydrophobic side-chains are still highly solvent accessible. A subsequently formed intermediate with helical structure in the alpha-domain is nearly identical in size and shape with native lysozyme and has a solvent-inaccessible hydrophobic core. Despite its native-like properties, this intermediate is only slightly more stable (DeltaG0=-4 kJ/mol) than the collapsed state and still much less stable than native lysozyme (DeltaDeltaG0=36 kJ/mol) at 20 degrees C.

Is cooperative oxygen binding by hemoglobin really understood?

The enormous success of structural biology challenges the physical scientist. Can biophysical studies provide a truly deeper understanding of how a protein works than can be obtained from static structures and qualitative analysis of biochemical data? We address this question in a case study by presenting the key concepts and experimental results that have led to our current understanding of cooperative oxygen binding by hemoglobin, the paradigm of structure function relations in multisubunit proteins. We conclude that the underlying simplicity of the two-state allosteric mechanism could not have been demonstrated without novel physical experiments and a rigorous quantitative analysis.

A statistical mechanical model for beta-hairpin kinetics.

Understanding the mechanism of protein secondary structure formation is an essential part of the protein-folding puzzle. Here, we describe a simple statistical mechanical model for the formation of a beta-hairpin, the minimal structural element of the antiparallel beta-pleated sheet. The model accurately describes the thermodynamic and kinetic behavior of a 16-residue, beta-hairpin-forming peptide, successfully explaining its two-state behavior and apparent negative activation energy for folding. The model classifies structures according to their backbone conformation, defined by 15 pairs of dihedral angles, and is further simplified by considering only the 120 structures with contiguous stretches of native pairs of backbone dihedral angles. This single sequence approximation is tested by comparison with a more complete model that includes the 2(15) possible conformations and 15 x 2(15) possible kinetic transitions. Finally, we use the model to predict the equilibrium unfolding curves and kinetics for several variants of the beta-hairpin peptide.

Folding dynamics and mechanism of beta-hairpin formation.

Protein chains coil into alpha-helices and beta-sheet structures. Knowing the timescales and mechanism of formation of these basic structural elements is essential for understanding how proteins fold. For the past 40 years, alpha-helix formation has been extensively investigated in synthetic and natural peptides, including by nanosecond kinetic studies. In contrast, the mechanism of formation of beta structures has not been studied experimentally. The minimal beta-structure element is the beta-hairpin, which is also the basic component of antiparallel beta-sheets. Here we use a nanosecond laser temperature-jump apparatus to study the kinetics of folding a beta-hairpin consisting of 16 amino-acid residues. Folding of the hairpin occurs in 6 micros at room temperature, which is about 30 times slower than the rate of alpha-helix formation. We have developed a simple statistical mechanical model that provides a structural explanation for this result. Our analysis also shows that folding of a beta-hairpin captures much of the basic physics of protein folding, including stabilization by hydrogen bonding and hydrophobic interactions, two-state behaviour, and a funnel-like, partially rugged energy landscape.

Laser temperature jump study of the helix<==>coil kinetics of an alanine peptide interpreted with a 'kinetic zipper' model.

The kinetics of the helix<==>coil transition of an alanine-based peptide following a laser-induced temperature jump were monitored by the fluorescence of an N-terminal probe, 4-(methylamino)benzoic acid (MABA). This probe forms a peptide hydrogen bond to the helix backbone, which changes its fluorescence quantum yield. The MABA fluorescence intensity decreases in a single exponential relaxation, with relaxation times that are weakly temperature dependent, exhibiting a maximum value of approximately 20 ns near the midpoint of the melting transition. We have developed a new model, the kinetic version of the equilibrium 'zipper' model for helix<==>coil transitions to explain these results. In this 'kinetic zipper' model, an enormous reduction in the number of possible species results from the assumption that each molecule contains either no helical residues or a single contiguous region of helix (the single-sequence approximation). The decay of the fraction of N-terminal residues that are helical, calculated from numerical solutions of the kinetic equations which describe the model, can be approximately described by two exponential relaxations having comparable amplitudes. The shorter relaxation time results from rapid unzipping (and zipping) of the helix ends in response to the temperature jump, while the longer relaxation time results from equilibration of helix-containing and non-helix-containing structures by passage over the nucleation free energy barrier. The decay of the average helix content is dominated by the slower process. The model therefore explains the experimental observation that relaxation for the N-terminal fluorescent probe is approximately 8-fold faster than that for the infrared probe of Williams et al. [(1996) Biochemistry 35, 691-697], which measures the average helix content, but does not account for the absence of observable amplitude for the slow relaxation in the fluorescence experiments (<10% slow phase). If we assume that the activation barrier for the coil-->helix rate is purely entropic, the model can also explain the maximum in the temperature dependence of the relaxation time for the fluorescent probe. Parameters that best reproduce the melting curves and the ratio of relaxation times predict a value of the cooperativity parameter sigma which is approximately 3-fold larger than previously reported values obtained from fitting equilibrium data only. The helix growth rate of approximately 10(8) s-1 that reproduces the experimental relaxation times is approximately 100-fold slower than those observed in molecular dynamics simulations. These parameters can be used to simulate the kinetically cooperative formation of a helix from the all-coil state.

Can a two-state MWC allosteric model explain hemoglobin kinetics?

We have analyzed the nanosecond-millisecond kinetics of ligand binding and conformational changes in hemoglobin. The kinetics were determined from measurements of precise time-resolved optical spectra following nanosecond photodissociation of the heme-carbon monoxide complex. To fit the data, it was necessary to extend the two-state allosteric model of Monod, Wyman, and Changeux (MWC) to include geminate ligand rebinding and nonexponential tertiary relaxation within the R quaternary structure. Considerable simplification of the model is obtained by using a linear free energy relation for the rates of quaternary transitions, and by incorporating concepts from recent studies on the physics of geminate rebinding and conformational changes in myoglobin. The model, described by 85 coupled differential equations, quantitatively explains a demanding set of complex kinetic data. Moreover, with the same set of kinetic parameters it simultaneously fits the equilibrium data on ligand binding and the distribution of ligation states. The present results, together with those from single-crystal oxygen binding studies, indicate that the two-state MWC allosteric model has survived its most critical tests.

Submillisecond protein folding kinetics studied by ultrarapid mixing.

An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent flow created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 micros to 3 ms. To eliminate the heme-ligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The fluorescence decrease is biphasic. There is an unresolved process with tau < 50 micros, followed by a slower, exponential process with tau = 600 micros at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.

Submillisecond kinetics of protein folding.

New experimental methods permit observation of protein folding and unfolding on the previously inaccessible nanosecond-microsecond timescale. These studies are beginning to establish times for the elementary motions in protein folding - secondary structure and loop formation, local hydrophobic collapse, and global collapse to the compact denatured state. They permit an estimate of about one microsecond for the shortest time in which a protein can possibly fold.

A multiparameter analysis of sickle erythrocytes in patients undergoing hydroxyurea therapy.

During 24 weeks of hydroxyurea treatment, we monitored red blood cell (RBC) parameters in three patients with sickle cell disease, including F-cell and F-reticulocyte profiles, distributions of delay times for intracellular polymerization, sickle erythrocyte adherence to human umbilical vein endothelial cells in a laminar flow chamber, RBC phthalate density profiles, mean corpuscular hemoglobin concentration and cation content, reticulocyte mean corpuscular hemoglobin concentration, 1H-nuclear magnetic resonance transverse relaxation rates of packed RBCs, and plasma membrane lateral and rotational mobilities of band 3 and glycophorins. Hydroxyurea increases the fraction of cells with sufficiently long delay times to escape the microcirculation before polymerization begins. Furthermore, high pretreatment adherence to human umbilical vein endothelial cells of sickle RBCs decreased to normal after only 2 weeks of hydroxyurea treatment, preceding the increase in fetal hemoglobin levels. The lower adhesion of sickle RBCs to endothelium would facilitate escape from the microcirculation before polymerization begins. Hydroxyurea shifted several biochemical and biophysical parameters of sickle erythrocytes toward values observed with hemoglobin SC disease, suggesting that hydroxyurea moderates sickle cell disease toward the milder, but still clinically significant, hemoglobin SC disease. The 50% reduction in sickle crises documented in the Multicenter Study of Hydroxyurea in Sickle Cell Disease is consistent with this degree of erythrocyte improvement.

Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding.

How fast can a protein fold? The rate of polypeptide collapse to a compact state sets an upper limit to the rate of folding. Collapse may in turn be limited by the rate of intrachain diffusion. To address this question, we have determined the rate at which two regions of an unfolded protein are brought into contact by diffusion. Our nanosecond-resolved spectroscopy shows that under strongly denaturing conditions, regions of unfolded cytochrome separated by approximately 50 residues diffuse together in 35-40 microseconds. This result leads to an estimate of approximately (1 microsecond)-1 as the upper limit for the rate of protein folding.

Fast events in protein folding.

Understanding how proteins fold is one of the central problems in biochemistry. A new generation of kinetic experiments has emerged to investigate the mechanisms of protein folding on the previously inaccessible submillisecond time scale. These experiments provide the first glimpse of processes such as secondary structure formation, local hydrophobic collapse, global collapse to compact denatured states, and fast barrier crossings to the native state. Key results are summarized and discussed in terms of the statistical energy landscape theory of protein folding.

A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity.

We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S-Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S-Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P < .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( > 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P < 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.