Metacognition of agency is reduced in high hypnotic suggestibility.

A disruption in the sense of agency is the primary phenomenological feature of response to hypnotic suggestions but its cognitive basis remains elusive. Here we tested the proposal that distorted volition during response to suggestions arises from poor metacognition pertaining to the sources of one's control. Highly suggestible and control participants completed a motor task in which performance was reduced through surreptitious manipulations of cursor lag and stimuli speed. Highly suggestible participants did not differ from controls in performance or metacognition of performance, but their sense of agency was less sensitive to cursor lag manipulations, suggesting reduced awareness that their control was being manipulated. These results indicate that highly suggestible individuals have aberrant metacognition of agency and may be a valuable population for studying distortions in the sense of agency.

Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease.

The design, synthesis, and biological evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Molecular-modeling experiments using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed us to design recognition features into the P1 and P2 subsites, respectively, from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addition, a series of ab initio calculations were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepared by general synthetic methods, starting with commercially available 5-substituted isatins in nearly every case. All compounds were tested for inhibition of purified HRV-14 3CP. Compounds 8, 14, and 19 were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compounds were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 A cocrystal structure of derivative 19 covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements.

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.

Efficacy against sheep lice (Bovicola ovis) and fleece wetting of six shower dip preparations.

The relative efficacy of 6 shower dip chemicals most frequently used for the treatment of sheep lice (Bovicola ovis) in Western Australia was examined. Groups of 20 sheep infested with lice were treated with products containing either alphamethrin, cyhalothrin, diazinon or diazinon plus piperonyl butoxide and rotenone, formulated as emulsifiable concentrates, and with products containing either coumaphos or magnesium fluorosilicate, formulated as wettable powders. All treatments were applied through a shower dip (Sunbeam model SSD). Inspections for lice were conducted until 9 months after dipping. No lice were found on sheep treated with the 4 emulsifiable concentrate products. In contrast, treatment with the wettable powders, which contained either coumaphos or magnesium fluorosilicate as the active ingredient, did not eradicate the lice infestations. The degree to which the fleece was wetted was assessed 20 minutes after dipping and showed that the wettable powder dips penetrated the fleece less than the emulsifiable concentrate dips. Less fluid was retained by wool staples in an in-vitro test when dip wash was made with the wettable powders. It was concluded that the degree of wetting attained at dipping was an important factor in achieving eradication of sheep lice.

Further evidence that zinc sulphate compromises the efficacy of dipping treatments using diazinon to control sheep lice (Bovicola ovis).

OBJECTIVE: To compare the wettability and efficacy of diazinon dip wash made with and without the addition of zinc sulphate. DESIGN: Field experiments using a shower and a plunge dip complemented by in-vitro wettability experiments. PROCEDURE: A flock of infested sheep was divided into groups and treated in a shower dip with clear or cloudy dam water plus up to 1.5% zinc sulphate. Another infested line of sheep was treated using a plunge dip with nil or 1% zinc sulphate. In both experiments, wetting was assessed after dipping and louse counts were conducted for 9 months after treatment. Five in-vitro experiments compared the wettability of dip wash containing diazinon with up to 1.5% zinc sulphate added. RESULTS: In the shower dipping experiment, live lice were found at 1 month after dipping in the cloudy water groups with 0.75%, 1.0% and 1.5% zinc sulphate and at 2 months in the 0.75% zinc sulphate group. No lice were found at subsequent inspections or at any time in the groups that were plunge dipped. Zinc sulphate decreased the amount of dip wash retained by wool staples in all in-vitro experiments (P < 0.05). CONCLUSION: Zinc sulphate should be considered as a risk factor that could cause failure to eradicate a lice infestation. The risk can be overcome by ensuring that all sheep are saturated at dipping and that the dip wash, and any holding tanks, are agitated throughout the dipping event.

Mortality of sheep exported by sea: evidence of similarity by farm group and of regional differences.

OBJECTIVE: To determine whether mortality of sheep exported by sea is similar for sheep from the same farm exported in different years or is associated with the region of origin. DESIGN: Mortalities were monitored in farm groups of sheep exported from the southwest of Western Australia under normal commercial conditions. PROCEDURE: Mortalities were monitored on commercial shipments from 1985 to 1996. For each consignment, the mortality rate was assigned its percentile ranking within the month and year of loading of the ship. A mortality rate was high if its percentile ranking was above a selected cut-off value. Five cut-off values were used in separate analyses. The spatial distribution of farms with high mortality was compared between and within zones of rainfall and length of pasture-growing season. RESULTS: A total of 479 groups of sheep from 405 farms was monitored. Mortality rates ranged from nil to 28.2%. Half of all deaths were from 14.2% of the consignments. There was a significant association (P < 0.05) between the category of mortality (high or low) in the first and second years of monitoring for four of the five cut-off values. The spatial analyses indicated that there were more high-mortality groups, and the average mortality was higher, in the zones of higher rainfall and longer pasture-growing season (P < 0.001). CONCLUSION: Mortality data can be used to identify regions and groups of sheep that are at risk of suffering high death rates when exported by sea.

A survey of ovine dermatophilosis in Western Australia.

A random sample of 200 Merino sheep owners was interviewed by telephone during April 1983 and asked questions relating to the prevalence of ovine dermatophilosis in their flocks, methods used for prevention and treatment of dermatophilosis, management strategies employed and the location and annual rainfall of each farm. The response rate was 99.5%. During the previous 12 months 62.3% of farmers had observed dermatophilosis in their flocks. The prevalence within flocks was highest in hoggets (mean 2.2%, range 0 to 75%) followed by lambs (mean 0.8%, range 0 to 25%), ewes (mean 0.6%, range 0 to 20%) and wethers (0.2%, range 0 to 20%). The mean weight of wool identified as affected by dermatophilosis was 58 kg (range 0 to 882 kg). Preventive measures were used on 57% of farms and the most common methods were changes in dipping practice (23.6%) and culling of affected sheep (21%). An average of 13.7 sheep per farm were culled for dermatophilosis and of these, 82% were sold and the remainder (18%) were killed on the farm. Antibiotics, of which most were combinations of penicillin and streptomycin were used to treat dermatophilosis on 8.5% of farms and treatments other than antibiotics were used on 10% of farms. The prevalence of dermatophilosis and its relationship to various environmental and management factors varied with the age and sex of sheep. Discriminant analysis indicated that of the factors studied, average annual rainfall, month of lambing, average fibre diameter and the month ewes were shorn were related to the prevalence of dermatophilosis in lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Mental health disaster response: nursing interventions across the life span.

In the aftermath of a natural disaster, adult survivors often move through the following phases of disaster response: Heroic Phase, Honeymoon Phase, Disillusionment Phase, and Reconstruction Phase. Understanding age-related responses to traumatic events such as a tornado enables mental health clinicians to individualize appropriate interventions and prevent or diminish emotional sequelae such as post-traumatic stress disorders. Psychiatric-mental health nurses are encouraged to attend local Red Cross disaster training to be prepared should the need arise for mobilization of mental health disaster response teams in your community.

The crystal structure of alpha-bungarotoxin at 2.5 A resolution: relation to solution structure and binding to acetylcholine receptor.

We report collection of 2.5 A resolution X-ray diffraction data from newly grown crystals of the rare 'small unit cell' form of the long snake neurotoxin, alpha-bungarotoxin. The previous model of the molecule has been rebuilt, and refined using least-square methods to a crystallographic residual of 0.24 at 2.5 A resolution. alpha-Bungarotoxin's crystal structure is compared with the crystal structures of two other snake neurotoxins (cobratoxin and erabutoxin), and with its solution structure inferred from spectroscopic studies. Significant differences include less beta-sheet in bungarotoxin's crystal structure than in solution, or in the crystal structures of other neurotoxins, and an unusual orientation in the crystal of the invariant tryptophan. The functional, binding surface of bungarotoxin is described; it consists primarily of hydrophobic and hydrogen-bonding groups and only a few charged side-chains. The structure is compared with experimental binding parameters for neurotoxins.

Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments.

We report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the 1H NMR spectrum of alpha-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper [Basus, V.J., & Scheek, R.M. (1988) Biochemistry (second paper of three in this issue)]. Significant differences exist between the solution structure described here and the crystal structure of alpha-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure [Love, R.A., & Stroud, R.M. (1986) Protein Eng. 1, 37]. These differences include a larger beta-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin alpha-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper [Kosen, P.A., Finer-Moore, J., McCarthy, M.P., & Basus, V.J. (1988) Biochemistry (third paper of three in this issue)].

Two telomerase reverse transcriptases (TERTs) expressed in Candida albicans.

The human pathogenic yeast Candida albicans contains two telomerase reverse transcriptase (TERT) genes. CaTERT1 and CaTERT2 appear either to be two alleles of the same gene or two entirely different genes that encode 867-residue proteins that differ by five amino acids. Both TERTs have a calculated pI of 9.5 and a M(r) of 100.9 kDa and are the smallest TERTs identified to date. Both genes appear to be expressed. Based on sequence similarity between CaTERT1 and the Saccharomyces cerevisiae orthologue Est2p, we suggest a revised alignment for motif E of Est2p. The identification of these TERT genes provides the first opportunity to study telomerase in an important human pathogen.

Subunit organization and structure of an acetylcholine receptor.

We have learned the positions of the alpha-subunits around the AChR rosette and the location of the toxin on the synaptic crest. A charge/hydrophobic character map of the 40 A X 30 A receptor surface that binds alpha-bungarotoxin has been constructed. A beta-structure domain surrounds the agonist binding site on the alpha-subunits, as predicted by amphipathic Fourier sequence analysis. The ion channel may be constructed from five amphipathic helices, which insert into the bilayer as the alpha2 beta gamma delta subunits come together. They form a water-filled channel on one side and interface with hydrophobic helices in each subunit on the other.

How the anti-(metal chelate) antibody CHA255 is specific for the metal ion of its antigen: X-ray structures for two Fab'/hapten complexes with different metals in the chelate.

Antibodies with bound metal-chelate haptens provide new means for exploiting the diverse properties of metallic elements. The murine monoclonal antibody CHA255 (IgG1 lambda) binds the metal-chelate hapten indium (III)-4-[N'-(2-hydroxyethyl)thioureido]-L-benzyl-EDTA (designated In-EOTUBE) with high affinity (K(a) = 1.1 x 10(10) M-1). Antibody binding is highly specific for the indium chelate; the affinity decreases as much as 10(4) with other metals, even those having ionic radii close to indium. To better understand this selectivity, the crystal structure of the antigen-binding fragment (Fab') of CHA255 complexed with its hapten, In(III)-EOTUBE, was determined by molecular replacement and refined at 2.2-A resolution. The structure of CHA255 Fab' complexed with Fe(III)-EOTUBE was also determined and refined at 2.8-A resolution. In both structures, the hapten's EDTA moiety is half-buried near the center of the complementarity-determining regions (CDR's). Five of the six CDR's on the Fab' interact with the hapten through protein side-chain atoms (but not main-chain atoms). A novel feature of the In-EOTUBE/Fab' complex is coordination of the indium by N epsilon of one histidine from the heavy chain's third CDR (distance = 2.4 A). The histidine coordination is not observed in the Fe-EOTUBE/Fab' complex, due mainly to a slightly different hapten conformation that reduces metal accessibility; this may partially explain the 20-fold lower affinity of CHA255 for iron hapten. An unexpected feature of the Fab' overall is an elbow angle of 193 degrees (the angle between the pseudodyad axes of the Fab's constant and variable domains).

The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.

The conformation of hepatitis C virus NS3 proteinase with and without NS4A: a structural basis for the activation of the enzyme by its cofactor.

BACKGROUND: Hepatitis C virus (HCV) NS3 proteinase activity is required for the release of HCV nonstructural proteins and is thus a potential antiviral target. The enzyme requires a protein cofactor NS4A, located downstream of NS3 on the polyprotein, for activation and efficient processing. OBJECTIVES: Comparison of the proteinase three-dimensional structure before and after NS4A binding should help to elucidate the mechanism of NS4A-dependent enzyme activation. STUDY DESIGN: We determined the crystal structure of NS3 proteinase of HCV BK isolate (genotype 1b; residues 1-189) and also the crystal structure of this proteinase complexed with HCV BK-NS4A (residues 21-34). RESULTS: The core region (residues 30-178) of the enzyme without cofactor (NS3P) or with bound cofactor (NS3P/4A) is folded into a trypsin-like conformation and the substrate P1 specificity pocket is essentially unchanged. However, the D1-E1 beta-loop shifts away from the cofactor binding site in NS3P/4A relative to NS3P, thereby accommodating NS4A. One result is that catalytic residues His-57 and Asp-81 move closer to Ser-139 and their sidechains adopt more 'traditional' (trypsin-like) orientation. The N-terminus (residues 1-30), while extended in NS3P, is folded into an alpha-helix and beta-strand that cover the bound cofactor of NS3P/4A. A new substrate-binding surface is formed from both the refolded N-terminus and NS4A, potentially affecting substrate residues immediately downstream of the cleavage site. CONCLUSIONS: Direct comparison of the crystal structures of NS3P and NS3P/4A shows that the binding of NS4A improves the anchoring and orientation of the enzyme's catalytic triad. This is consistent with the enhancement of NS3P's weak residual activity upon NS4A binding. There is also significant refolding of the enzyme's N-terminus which provides new interactions with P'-side substrate residues. The binding surface for P'-side substrate residues, including the P1 specificity pocket, changes little after NS4A binding. In summary, we observe a structural basis for improved substrate turnover and affinity that follows complexation of NS3P with its NS4A cofactor.

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.