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.

Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase.

Several proteins have been discovered that either catalyze slow protein-folding reactions or assist folding in the cell. Prolyl isomerase, which has been shown to accelerate rate-limiting cis-trans peptidyl-proline isomerization steps in the folding pathway, can also participate in the protein-folding process as a chaperone. This function is exerted on an early folding intermediate of carbonic anhydrase, which is thereby prevented from aggregating, whereas the isomerase activity is performed later in the folding process.

Folding of beta-sheet proteins.

In the past year, interesting new information concerning various aspects of the folding process of beta-sheet proteins has been gleaned. Kinetic and equilibrium folding intermediates have been characterized. Studies of extensively denatured states and of model peptide fragments have enabled important steps to be taken towards an understanding of the initiation of the folding process of beta-sheet proteins. Site-directed mutagenesis has been used in combination with various probes to monitor folding events.

The role of the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II.

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.

The existence of glutathione and cysteine disulfide-linked to erythrocyte carbonic anhydrase from tiger shark.

In this study it is shown that the higher molecular weight previously reported for tiger shark carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) compared to other carbonic anhydrases is decreased to a normal value around 30 000 after disulfide reduction of the enzyme. This difference in molecular weight is at least partly due to the existence of disulfide-linked glutathione and cysteine residues. Approx. 3 mol glutathione and a similar amount of cysteine are shown to be bound per mol enzyme. The presence of these factors also has effects on the enzyme activity.

Purification and properties of cyclostome carbonic anhydrase from erythrocytes of hagfish.

1. Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified from erythrocytes of hagfish (Myxine glutinosa). A single form with low specific CO2 hydration activity was isolated. The purified carbonic anhydrase appeared homogeneous judging from polyacrylamide gel electrophoresis and gel filtration experiments. The protein has a molecular weight of about 29 000, corresponding to about 260 amino acid residues. This molecular weight is in accordance with other vertebrate carbonic anhydrases with the exception of the elasmobranch enzymes, which have Mr 36 000--39 000. 2. The molecular weight obtained for hagfish carbonic anhydrase indicates that a carbonic anhydrase with Mr approx. 29 000 is the ancestral type of the vertebrate enzyme rather than, as in sharks, a heavier carbonic anhydrase molecule. 3. The circular dichroism spectrum may indicate a somewhat different structural arrangement of aromatic amino acid residues in this enzyme than in the mammalian carbonic anhydrases. 4. The enzyme is strongly inhibited by acetazolamide and also to a lesser extent by monovalent anions. 5. Zn2+, which is essential for activity, appears, contrary to other characterized carbonic anhydrases, less strongly bound in the active site of the enzyme.

Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum.

Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes.

GroEL reversibly binds to, and causes rapid inactivation of, human carbonic anhydrase II at high temperatures.

The initial yield of reactivation of GuHCl denatured human carbonic anhydrase II does not change with temperature between 3 and 35 degrees C. At temperatures above 35 degrees C, the enzymatic activity is not stable, but decreases over time. If the bacterial chaperonin GroEL is present during reactivation, the initial yield is lower compared to the spontaneous reaction at temperatures of 35-50 degrees C. However, unlike the spontaneous reactivation, the enzymatic activity with time in the presence of GroEL. In the presence of GroEL, native HCA II incubated at elevated temperatures will rapidly loose enzymatic activity to the same value as during reactivation at that particular temperature; most of the activity will recover if the temperature is lowered when GroEL is present. It is evident that there is an equilibrium between an inactive intermediate of HCA II, probably bound to GroEL, and active enzyme. Furthermore, proline isomerization is part of the rate-limiting step of refolding even in the presence of GroEL, and it is very noteworthy that prolyl isomerase will influence the refolding of HCA II in the presence of GroEL.

Role of an evolutionarily invariant serine for the stability of human carbonic anhydrase II.

There are several evolutionarily invariant amino acids in the primary structures of all known isoenzymes of carbonic anhydrase. One of these is Ser-29 which is situated in the peripheral part of the active site interacting by hydrogen bonds with amino acids located nearby in the tertiary structure. Furthermore, the neighbourhood of Ser-29, composed of Gln-28, Pro-30, Tyr-194, Ser-197 and Trp-209, has a totally invariant structure. The structural role of Ser-29 was investigated by site-directed mutagenesis. The stability of two enzyme mutants, where Ser-29 was replaced by alanine and cysteine, towards denaturation by guanidine-HCl was studied. Changing Ser-29 to Ala resulted in a destabilization by 2.6 kcal/mol, corresponding to the loss of 2-3 hydrogen bonds. Interestingly, Ser-29 is within hydrogen bond distance to Tyr-194, Ser-197 and Trp-209 in the tertiary structure. Therefore, rupture of these interactions caused by the Ser-29----Ala substitution could explain the observed destabilization of this enzyme variant. Substituting cysteine for Ser-29 gives rise to a drastic decrease in the stability of the protein (change in midpoint concentration of denaturation from 0.96 M to less than 0.1 M guanidine-HCl) despite the minor structural change (O----S atom). This destabilization corresponds to approx. 7-8 kcal/mol and cannot be explained by changes in hydrogen bond pattern only, but must also include unfavourable conformational changes to avoid van der Waals collisions originating from the somewhat larger thiol group.

GroEL/ES-mediated refolding of human carbonic anhydrase II: role of N-terminal helices as recognition motifs for GroEL.

The presence of GroEL/ES during the refolding of human carbonic anhydrase II (pseudo-wild type) was found to increase the yield of active enzyme from 65 to 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time-course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonic anhydrase, in which N-terminal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: impact on calculation of secondary structure content.

The CD spectra of human carbonic anhydrase I and II and bovine carbonic anhydrase III were recorded and analyzed. The 3D structures of these isoenzymes are known, showing very similar secondary structure and polypeptide-chain fold. The tryptophan content, however, differs between the isoenzymes, i.e., isoenzymes I, II, and III possess 6, 7, and 8 tryptophans, respectively. All of the tryptophans except the additional tryptophans in isoenzymes II and III, i.e., W245 and W47, are conserved. Despite the fact that X-ray structure determinations showed that the isoenzymes had highly similar secondary structure, the contents of alpha-helix and beta-sheet structure differed considerably when using different CD algorithms for estimation of the fractions of various secondary structural elements. This shows that aromatic amino acids also interfere in the wavelength region (far-UV) used to calculate the amount of secondary structure. Such interference is especially problematic when analyzing proteins like carbonic anhydrase, which consist mainly of beta-structure that gives rise to weak ellipticity bands, compared to the bands arising from alpha-helical structure.

GroEL provides a folding pathway with lower apparent activation energy compared to spontaneous refolding of human carbonic anhydrase II.

The kinetics of the refolding of the enzyme, human carbonic anhydrase II (HCA II), at different temperatures, together with the Escherichia coli chaperonin GroEL, has been studied. The Arrhenius plots for the spontaneous, GroEL-assisted, and GroEL/ES-assisted refolding of HCA II show that the apparent activation energy (E(a)) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES-assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES-assisted reactions, respectively).

Folding around the C-terminus of human carbonic anhydrase II. Kinetic characterization by use of a chemically reactive SH-group introduced by protein engineering.

We are characterizing the process of refolding of the enzyme human carbonic anhydrase II from the denatured state in guanidine hydrochloride. To describe the folding in defined parts of the protein we use protein engineering to introduce cysteine residues as unique chemically reactive probes. The accessibility of the cysteine SH-group to the alkylating reagent iodoacetate, at different stages during refolding, is used to give a kinetic description of the folding process. The structuration of the C-terminal part of the polypeptide chain, which is involved in a unique 'knot' topology, was investigated. Our results show that the structure around the C-terminal, composed of the outermost beta-strands in a dominating beta-structure that extends through the entire protein, is formed relatively late during refolding. In contrast, it was found that beta-strands located in the interior of the protein were structured very rapidly. The final native structure is formed in a process that is slower than those observed for formation of beta-structure.

Pyrene excimer fluorescence as a proximity probe for investigation of residual structure in the unfolded state of human carbonic anhydrase II.

The excimer fluorescence from two pyrenyl moieties attached to cysteines in human carbonic anhydrase II has been monitored to characterize residual structure retained under strong denaturing conditions. A position in beta-strand 3, N67C, together with the single naturally occurring cysteine 206 in beta-strand 7, were used as attachment sites. The eximer formation by the pyrenyls, requiring proximity of the probes, revealed an unfolding transition at a GuHCl concentration significantly higher than that required to induce unfolding of the molten globule state as monitored by CD. These results indicate that the excimer transition monitors the unfolding of a residual compact structure that spans beta-strands 3-7. This region constitutes the central and the most hydrophobic part of the molecule, emphasizing the importance of hydrophobic interaction in maintaining residual structure under strong unfolding conditions.

Lack of correspondence between the room-temperature phosphorescence decay-components and Trp residues in a series of Trp-->Cys or Trp-->Phe mutants of human carbonic anhydrase II.

The room temperature phosphorescence of native human carbonic anhydrase (CA), and several mutants of this enzyme has been investigated. In these mutants the seven tryptophan residues in the native protein have sequentially been replaced by cysteine or phenylalanine. All of the mutants as well as native CA show room-temperature tryptophan phosphorescence (RTP) spectra. Surprisingly, only small differences in RTP life-times are noticeable among these mutants, indicating that there is more than one tryptophan residue with similar phosphorescence decay kinetics in the protein. The present results illustrate the danger in attributing the room temperature phosphorescence of a multi-tryptophan protein to a particular residue based solely on an analysis of the protein structure.

Adsorption to silica nanoparticles of human carbonic anhydrase II and truncated forms induce a molten-globule-like structure.

Human carbonic anhydrase II pseudo-wild type (HCAIIpwt) and two truncated variants were adsorbed to approximately 9 nm silica nanoparticles. Ellipsometry was used as an indirect measure of protein adsorption. The structural changes of adsorbed proteins were investigated with the use of circular dichroism (CD), intrinsic fluorescence, ANS binding ability and inhibitor binding capacity. It was found that the variants that were truncated at positions 5 and 17 in the N-terminal end attain a molten-globule-like state after interaction with the silica nanoparticles. In contrast, the more stable HCAIIpwt retained most of its native structure after 24 h adsorption to silica nanoparticles. The result suggests that surface induced unfolding may give rise to intermediates similar to those for unfolding induced by, for example GuHCl. Thus, the intermediate observed has some features of the molten globule.

Cis-trans isomerization is rate-determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase.

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t1/2 = 9 min). Kinetic data from 'double-jump' measurements indicate that proline isomerization might be rate determining in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics: the presence of isomerase during reactivation lowers the half-time of the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis-peptidyl-Pro bonds at Pro30 and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) were constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPIase the P202N mutant behaved exactly like the unmutated enzyme. Thus, cis-trans isomerization of the Pro202 cis-peptidyl bond is not rate determining in the reactivation process. The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.

The adenoma-carcinoma sequence in rectal adenomas. Support by the expression of blood group substances and carcinoma antigens.

The reaction patterns of eight antibodies directed against blood group substances A, B, and H and against Lewis B antigen, difucosylated carbohydrate antigens (DFCA), gastrointestinal cancer antigen (GICA) CA 19-9, carcinoma-associated antigen CA-50, and carcinoembryonic antigen (CEA) were studied in mucosa and adenomas of the rectum. Antigenic heterogeneity was a common feature of rectal mucosa and was observed to a considerable degree in adenomas. In general, the rectal mucosa expressed only a few antigens and to a limited extent. The adenomas were more extensively stained than the rectal mucosa. The proportion of positive cells increased with the grade of dysplasia but was relatively unrelated to the histologic type. The proportion of individual antigens expressed and the number of antigens extensively expressed increased between rectal mucosa, benign adenomas, and adenomas with early invasive carcinoma. These findings support the concept of an adenoma (dysplasia)-carcinoma sequence.

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.

Effect of bovine virus diarrhoea virus on pregnancy in the ewe monitored by ultrasound scanning and levels of 15-keto-13,14-dihydro-PGF2 alpha and progesterone.

Fourteen unvaccinated and seronegative ewes were experimentally infected with bovine virus diarrhoea virus around day 60 of pregnancy. Two other groups of pregnant ewes served as control animals. The animals were followed with ultrasound scanning to study the status of the fetus and with frequent blood sampling for analysis of progesterone and 15-keto-13,14-dihydro-PGF2 alpha, two hormones that could reflect the propagation of the infection. The unvaccinated ewes responded to the infection with abortion, resorption of the fetus, mummification or no changes at all. The endocrinological changes reflected in a most adequate way the pathological changes in the uterus. Detailed endocrinological studies can contribute to the understanding of the pathogenesis of some infectious diseases that affect reproduction.