Investigations and actions taken during 2011 due to the first finding of Echinococcus multilocularis in Sweden.

Echinococcus multilocularis is a parasite that can cause alveolar echinococcosis disease. After the first positive finding of E. multilocularis in Sweden in 2011, a consulting group with representatives from relevant authorities was summoned. In this group, all relevant information was shared, strategies for information dissemination and any actions to be taken due to the finding of E. multilocularis were discussed and decided. The present paper describes the actions taken during 2011 and the results thereof, including surveillance in animals, risk assessment for humans to become infected and recommendations given to the public. Further discussion about whether the parasite was introduced, and if so, how, as well as possible future development of the infection in animals and humans in Sweden and future actions are included.

Global animal disease surveillance.

Development and implementation of global animal disease surveillance has been limited by the lack of information systems that enable near real-time data capturing, sharing, analysis, and related decision- and policy-making. The objective of this paper is to describe requirements for global animal disease surveillance, including design and functionality of tools and methods for visualization and analysis of animal disease data. The paper also explores the potential application of techniques for spatial and spatio-temporal analysis on global animal disease surveillance, including for example, landscape genetics, social network analysis, and Bayesian modeling. Finally, highly pathogenic avian influenza data from Denmark and Sweden are used to illustrate the potential application of a novel system (Disease BioPortal) for data sharing, visualization, and analysis for regional and global surveillance efforts.

Emergence of porcine reproductive and respiratory syndrome in Sweden: detection, response and eradication.

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sows and respiratory problems in growing pigs. The disease is present in most countries throughout the world but was not diagnosed in Sweden until the summer of 2007 when it was first detected through the national PRRS surveillance program. The immediate mobilization of veterinary authorities, field veterinarians and the pig industry was a prerequisite for preventing the spread of the disease. Within 10 days seven herds were verified as infected and the measures taken included stamping out, cleaning, disinfection and a vacancy period of 3 weeks before the herds were repopulated. To evaluate the effectiveness of these measures, a national sero-surveillance was carried out during the autumn of 2007. Approximately 90% of the pig production was covered by this screening and all samples tested were negative with regard to antibodies to PRRS virus.

A conformationally isoformic thermophilic protein with high kinetic unfolding barriers.

The basis for the stability of thermophilic proteins is of fundamental interest for extremophile biology. We investigated the folding and unfolding processes of the homotetrameric Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). TBADH subunits were 4.8 kcal/mol less stable towards guanidinium chloride (GdmCl) unfolding compared to urea, indicating ionic modulation of TBADH stability. Strongly denaturing conditions promoted mono-exponential unfolding kinetics with linear dependence on denaturant concentration. Here TBADH unfolded >40-fold slower when extrapolated from urea as compared to GdmCl unfolding. A marked unfolding hysteresis was shown when comparing refolding and unfolding in urea. An unusual biphasic unfolding trajectory with an exceptionally slow phase at intermediate concentrations of GdmCl and urea was also observed. We advocate that TBADH forms two distinctly different tetrameric isoforms, and likely an ensemble of native states. This unusual supramolecular folding behavior has been shown responsible for formation of amyloidotic yeast prion strains and can have functional importance for TBADH.

The acute phase response in calves experimentally infected with bovine viral diarrhoea virus and/or Mannheimia haemolytica.

The aim of this investigation was to study differences and similarities in the acute phase response of calves experimentally infected in the respiratory tract with either bovine viral diarrhoea virus (BVDV) or Mannheima haemolytica (Mh), or with a combination of both (BVDV/Mh). A non-inoculated control group was also included. The acute phase response was measured by serum or plasma concentrations of the acute phase proteins (APPs) haptoglobin, serum amyloid A (SAA) and fibrinogen, and of cortisol, prostaglandin F2alpha-metabolite and interferon-alpha (IFN-alpha) activity. Clinical symptoms were also recorded and were most severe in the BVDV/Mh group. The symptoms were mild to moderate in the BVDV group, while none, or very mild symptoms were observed in the Mh group. In all inoculated groups, a significant acute phase response was observed, with elevated values of haptoglobin, SAA and fibrinogen, while the control group remained unaffected throughout the study. In general, the magnitude of the response was similar, but the duration of elevated concentrations of APPs was significantly longer in the BVDV/Mh group than in the BVDV group, reflecting the duration of the clinical symptoms. However, in the single infection groups, the APP response and the clinical symptoms were not correlated. The IFN-alpha activity increased in all BVDV-inoculated animals, but no response in cortisol and PGF2alpha-metabolite concentrations was observed after infection. Basal levels of serum concentrations of haptoglobin, SAA and fibrinogen were established and may be used for evaluating calf health in herds. The duration of elevated haptoglobin, SAA and fibrinogen values did not differ significantly within groups indicating that their value as indicator of disease is equal.

Spin and fluorescent probing of the binding interface between tissue factor and factor VIIa at multiple sites.

The specific complex between the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVIIa interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma-carboxyglutamic acid (Gla) domain of FVIIa, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVIIa. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVIIa binding, especially in the sTF-Gla region. There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVIIa, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.

Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor.

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s(-1). For the mutants, dissociation rates were faster (0.022-0.025 s(-1)), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa.

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF--FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II.

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.

High-resolution probing of local conformational changes in proteins by the use of multiple labeling: unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes.

Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)-ethyl)amino)naphtalene-1-sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation. HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 A from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 A from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At approximately 1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 A in diameter depending on the experimental conditions and spectroscopic technique used.

Cofactor-induced refolding: refolding of molten globule carbonic anhydrase induced by Zn(II) and Co(II).

The stability versus unfolding to the molten globule intermediate of bovine carbonic anhydrase II (BCA II) in guanidine hydrochloride (GuHCl) was found to depend on the metal ion cofactor [Zn(II) or Co(II)], and the apoenzyme was observed to be least stable. Therefore, it was possible to find a denaturant concentration (1.2 M GuHCl) at which refolding from the molten globule to the native state could be initiated merely by adding the metal ion to the apo molten globule. Thus, refolding could be performed without changing the concentration of the denaturant. The molten globule intermediate of BCA II could still bind the metal cofactor. Cofactor-effected refolding from the molten globule to the native state can be summarized as follows: (1) initially, the metal ion binds to the molten globule; (2) compaction of the metal-binding site region is then induced by the metal ion binding; (3) a functioning active center is formed; and (4) finally, the native tertiary structure is generated in the outer parts of the protein.

Protein compactness measured by fluorescence resonance energy transfer. Human carbonic anhydrase ii is considerably expanded by the interaction of GroEL.

Nine single-cysteine mutants were labeled with 5-(2-iodoacetylaminoethylamino)naphthalene-1-sulfonic acid, an efficient acceptor of Trp fluorescence in fluorescence resonance energy transfer. The ratio between the fluorescence intensity of the 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid (AEDANS) moiety excited at 295 nm (Trp absorption) and 350 nm (direct AEDANS absorption) was used to estimate the average distances between the seven Trp residues in human carbonic anhydrase II (HCA II) and the AEDANS label. Guanidine HCl denaturation of the HCA II variants was also performed to obtain a curve that reflected the compactness of the protein at various stages of the unfolding, which could serve as a scale of the expansion of the protein. This approach was developed in this study and was used to estimate the compactness of HCA II during heat denaturation and interaction with GroEL. It was shown that thermally induced unfolding of HCA II proceeded only to the molten globule state. Reaching this state was sufficient to allow HCA II to bind to GroEL, and the volume of the molten globule intermediate increased approximately 2.2-fold compared with that of the native state. GroEL-bound HCA II expands to a volume three to four times that of the native state (to approximately 117,000 A(3)), which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity. Recently, we found that HCA II binding causes such an inflation of the GroEL molecule, and this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (Hammarström, P., Persson, M., Owenius, R., Lindgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838), thereby facilitating rearrangement of misfolded structure.

Contribution of tryptophan residues to the CD spectrum of the extracellular domain of human tissue factor: application in folding studies and prediction of secondary structure.

The contribution to the circular dichroism (CD) spectrum made by each of the four Trp residues in the extracellular domain of human tissue factor, sTF (s designates soluble), was determined from difference CD spectra. The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated (180-305 nm). The sum of the individual spectra of each Trp residue in the near-UV region was qualitatively identical to the wild-type spectrum, clearly demonstrating that the Trp residues are the major contributors to the spectrum in this wavelength region. Trp CD bands interfere with the peptide bands in the far-UV region, leading to uncertainty in the predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp-free CD spectrum obtained after subtraction of all individual Trp spectra from the wild-type spectrum. The mutated Trp residues were also exploited as intrinsic probes to monitor the formation of local native-like tertiary structure by kinetic near-UV CD measurements. The global folding reaction was followed in parallel with a novel functional assay that registered the recovery of cofactor activity, i.e. stimulation of the amidolytic activity of Factor VIIa. From these measurements, it was found that sTF appears to regain FVIIa cofactor activity before the final side-chain packing of the Trp residues. The combined kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.

Plantar flexor muscle function in open and closed chain.

In the present study, the torque or work produced during isometric, pure concentric and eccentric-concentric plantar flexions, performed in sitting, standing and prone were measured. The electromyographic (EMG) activity was measured from the soleus, gastrocnemius medialis, tibialis anterior and rectus femoris muscles. The isometric tests showed the highest torques in the standing test. The rectus femoris and gastrocnemius activities were lower in the prone than in the standing test. The sitting test showed lower activities in all muscles of the lower leg compared with the standing test. No differences in work between the prone and sitting tests were found during the concentric phases. Higher rectus femoris activity in the eccentric-concentric test and lower activity in the triceps surae during the concentric phases were seen in the sitting compared with the prone test. We conclude that tests of overall functional ability should be performed in the standing position while specific tests of the plantar flexors should be performed in the prone position.

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.

Localization of the Cl-/HCO3- anion exchanger binding site to the amino-terminal region of carbonic anhydrase II.

Human carbonic anhydrase II (CAII) possesses a binding site for an acidic motif (D887ADD) within the carboxyl-terminal region (Ct) of the human erythrocyte chloride/bicarbonate anion exchanger, AE1. In this study, the amino acid sequence comprising this AE1 binding site was localized to the first 17 residues of CAII, which form a basic patch on the surface of the protein. Truncation of the amino terminal of CAII by five residues resulted in a 3-fold reduction in the apparent affinity of the interaction with a GST fusion protein of the Ct of AE1 (GST-Ct) measured by a sensitive microtiter plate binding assay. Further amino-terminal truncation of CAII by 17 or 24 residues caused a loss of binding. The homologous isoform CAI does not bind AE1, despite having 60% sequence identity to CAII. One major difference between the two CA isoforms, within the amino-terminal region, is a high content of histidine residues in CAII (His3, -4, -10, -15, -17) not found in CAI. Mutation of pairs of these histidines (and one lysine) in CAII to the analogous residues in CAI (H3P/H4D or K9D/H10K or H15Q/H17S), or combinations of these various double mutants, did not greatly affect binding between GST-Ct and the mutant CAII. However, when all six of the targeted CAII residues were mutated to the corresponding sequence in CAI, binding of GST-Ct was lost. These results indicate that the AE1 binding site is located within the first 17 residues of CAII, and that the interaction is mediated by electrostatic interactions involving histidine and/or lysine residues. Further specificity for the interaction of AE1 and CAII is provided by a conserved leucine residue (L886) in AE1 that, when mutated to alanine, resulted in loss of GST-Ct binding to immobilized CAII. The binding of the basic amino-terminal region of CAII to an acidic Ct in AE1 provides a structural basis for linking bicarbonate transport across the cell membrane to intracellular bicarbonate metabolism.

Postural disturbance in patients with normal pressure hydrocephalus.

The postural function in 52 patients with normal pressure hydrocephalus (NPH) and in 19 patients with subcortical arteriosclerotic encephalopathy (SAE) was analysed bedside and in 17 of the NPH, 10 of the SAE and 23 healthy individuals (HI) also examined with a force platform. At the bedside examination, no differences in postural functions between NPH and SAE patients were found. The NPH patients improved more in the postural than in motor functions after shunt surgery. The NPH patients had a larger sway area and a higher backward directed velocity of centre of pressure than HI. The direction of the inclination in the sagittal plane was neutral or forward in the NPH and the SAE patients while it was backward in HI. The postural function was better in positions with open eyes in all 3 groups, but significantly less in the NPH patients, indicating a misinterpretation of afferent visual stimuli in the brainstem postural centre.

Is the unfolded state the Rosetta Stone of the protein folding problem?

Solving the protein folding problem is one of the most challenging tasks in the post genomic era. Identification of folding-initiation sites is very important in order to understand the protein folding mechanism. Detection of residual structure in unfolded proteins can yield important clues to the initiation sites in protein folding. A substantial number of studied proteins possess residual structure in hydrophobic regions clustered together in the protein core. These stable structures can work as seeds in the folding process. In addition, local preferences for secondary structure in the form of turns for beta-sheet initiation and helical turns for alpha-helix formation can guide the folding reaction. In this respect the unfolded states, studied at increasing structural resolution, can be the Rosetta Stone of the protein folding problem.