Experimentally induced and real-world anxiety have no demonstrable effect on goal-directed behaviour.

BACKGROUND: Goal-directed control guides optimal decision-making and it is an important cognitive faculty that protects against developing habits. Previous studies have found some evidence of goal-directed deficits when healthy individuals are stressed, and in psychiatric conditions characterised by compulsive behaviours and anxiety. Here, we tested if goal-directed control is affected by state anxiety, which might explain the former results. METHODS: We carried out a causal test of this hypothesis in two experiments (between-subject N = 88; within-subject N = 50) that used the inhalation of hypercapnic gas (7.5% CO2) to induce an acute state of anxiety in healthy volunteers. In a third experiment (N = 1413), we used a correlational design to test if real-life anxiety-provoking events (panic attacks, stressful events) are associated with impaired goal-directed control. RESULTS: In the former two causal experiments, we induced a profoundly anxious state, both physiologically and psychologically, but this did not affect goal-directed performance. In the third, correlational, study, we found no evidence for an association between goal-directed control, panic attacks or stressful life eventsover and above variance accounted for by trait differences in compulsivity. CONCLUSIONS: In sum, three complementary experiments found no evidence that anxiety impairs goal-directed control in human subjects.

Dispersing artifacts in FT-STS: a comparison of set point effects across acquisition modes.

Fourier-transform scanning tunnelling spectroscopy (FT-STS), or quasiparticle interference, has become an influential tool for the study of a wide range of important materials in condensed matter physics. However, FT-STS in complex materials is often challenging to interpret, requiring significant theoretical input in many cases, making it crucial to understand potential artifacts of the measurement. Here, we compare the most common modes of acquiring FT-STS data and show through both experiment and simulations that artifact features can arise that depend on how the tip height is stabilized throughout the course of the measurement. The most dramatic effect occurs when a series of dI/dV maps at different energies are acquired with simultaneous constant current feedback; here a feature that disperses in energy appears that is not observed in other measurement modes. Such artifact features are similar to those arising from real physical processes in the sample and are susceptible to misinterpretation.

Quantifying many-body effects by high-resolution Fourier transform scanning tunneling spectroscopy.

High-resolution Fourier transform scanning tunneling spectroscopy (FT-STS) is used to study many-body effects on the surface state of Ag(111). Our results reveal a kink in the otherwise parabolic band dispersion of the surface electrons and an increase in the quasiparticle lifetime near the Fermi energy Ef. The experimental data are accurately modeled with the T-matrix formalism for scattering from a single impurity, assuming that the surface electrons are dressed by the electron-electron and electron-phonon interactions. We confirm the latter as the interaction responsible for the deviations from bare dispersion. We further show how FT-STS can be used to simultaneously extract real and imaginary parts of the self-energy for both occupied and unoccupied states with a momentum and energy resolution competitive with angle-resolved photoemission spectroscopy. From our quantitative analysis of the data we extract a Debye energy of ℏΩD=14±1 meV and an electron-phonon coupling strength of λ=0.13±0.02, consistent with previous results. This proof-of-principle measurement advances FT-STS as a method for probing many body effects, which give rise to a rich array of material properties.

Scanning tunneling spectroscopy of superconducting LiFeAs single crystals: evidence for two nodeless energy gaps and coupling to a bosonic mode.

The superconducting compound LiFeAs is studied by scanning tunneling microscopy and spectroscopy. A gap map of the unreconstructed surface indicates a high degree of homogeneity in this system. Spectra at 2 K show two nodeless superconducting gaps with Δ(1)=5.3±0.1 meV and Δ(2)=2.5±0.2 meV. The gaps close as the temperature is increased to the bulk T(c), indicating that the surface accurately represents the bulk. A dip-hump structure is observed below T(c) with an energy scale consistent with a magnetic resonance recently reported by inelastic neutron scattering.

Versican overexpression in cutaneous malignant melanoma.

OBJECTIVE: Tumor growth regulation by extracellular matrix components has been one of the main topics on tumor biology in the last years. We aimed to investigate the protein expression pattern of decorin and versican in superficial spreading melanoma (SSM) and its precursors. PATIENTS AND METHODS: Paraffin-embedded sections of benign nevi (BN), dysplastic nevi (DN), and primary SSM were assessed. Immunohistochemistry was performed for decorin and versican antibodies. RESULTS: We investigated 64 patients with BN (n = 29), DN (n = 15), and SSM (n = 20) with a median Breslow thickness of 0.8 mm (0.2 - 4.6 mm). We did not observe decorin or versican immunoreactivity in melanocytes but in peritumoral stroma. Kruskal-Wallis ANOVA did not reveal significant differences of decorin expression between the groups investigated (P = 0.19). However, compared to BN and DN median expression of versican was significantly increased in SSM (P = 0.016 and P = 0.019, respectively). Decorin as well versican expression of SSM did not significantly correlate with Breslow tumor thickness or Clark level. CONCLUSION: Our data indicate that decorin is not differentially expressed in peritumoral stroma of SSM, DN, BN, and thus unlikely of pathogenetic significance in melanoma transformation and/or progression. By contrast, we have demonstrated that SSM is associated with a significant overexpression of peritumoral versican suggesting a role for versican in the pathogenesis of melanoma.

A fusion plasmid for the synthesis of lipopeptide-antigen chimeras in Escherichia coli.

Lipopeptides are potential vaccine candidates with a built-in adjuvant property. To circumvent the present chemical route of synthesis for lipopeptide-antigen conjugates, the lipoprotein property of the pColE2-P9-encoded lysis protein, CelB, was used to create the bacterial fusion plasmid, pKLY3, to produce lipopeptide-antigen chimeras in Escherichia coli. Plasmid pKLY3 is a derivative of pKK233-2 with the origin of replication of the single-stranded DNA phage, fl. Under control of the promoter, ptrc, is the 5' end of the celB gene coding for a lipoprotein signal peptide and the first five amino acids (aa) (CQANY) of the mature lysis protein. As model systems for the synthesis of small and large lipopeptide-antigens, DNA sequences coding for the P2 peptide and E. coli alkaline phosphatase (PhoA) were fused in frame to the region of celB coding for a lipoprotein signal peptide and CQANY. P2 is a 12-aa peptide including a tyrosine phosphorylation site of the epidermal growth factor receptor (EGF-R). Inducible expression of stable lipohexapeptide CQANYV, lipo-CQANY-P2, and lipo-CQANYA-PhoA, was demonstrated. Similar expression was obtained for lipo-CIEGR-P2 and lipo-CIEGRA-PhoA in which IEGR is a cleavage recognition site for the blood coagulation factor, Xa. Like QANY, IEGR is predicted to form a beta-turn structure. The presence of a lipid moiety on the products was confirmed by demonstrating the incorporation of radioactive palmitic acid and inhibition of processing by globomycin. The lipid-modified peptides were also identified by incorporation of radioactive tyrosine, and the nature of the P2 peptide was verified immunologically.(ABSTRACT TRUNCATED AT 250 WORDS)

Two proline porters in Escherichia coli K-12.

Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus.

Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift.

Proline transport and osmotic stress response in Escherichia coli K-12.

Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.

NMR study of the differential contributions of residues of transforming growth factor alpha to association with its receptor.

A heteronuclear NMR study of human transforming growth factor alpha (TGFalpha) in complex with the epidermal growth factor receptor extracellular domain (EGFR-ED) provided an effective method for delineating the relative contributions of the residues of the ligand to its affinity for the receptor. In conjunction with previously obtained mutagenesis data, these results indicate that while a large number of residues are involved in complex formation and make up the binding interface, a small subset contribute most of the binding energy. They also show that while the residues which contribute to receptor binding are localized on one face of the molecule, the specific residues that play the major role in the affinity of TGFalpha in the complex are in two distinct regions of TGFalpha. This suggests that two small functional epitopes each composed of two residues exist within a larger structural epitope presented on the binding face. These results give the most detailed picture to date of the receptor binding determinants and yield further insight into the formation of the ligand-receptor complex.

Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta ) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding.

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-beta can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-beta isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-beta molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.

Evidence of a major neutralizable conformational epitope region on VP2 of infectious pancreatic necrosis virus.

A collection of neutralizing monoclonal antibodies (MAbs) produced against the LWVRT 60.1, Jasper and N1 strains of infectious pancreatic necrosis virus (IPNV) were selected for the analysis of VP2 epitopes. Previous characterization of the LW and JA MAbs allowed the identification of continuous and discontinuous epitopes but the topological localization of these sites remained obscure. The ability of these MAbs to differentiate individual epitopes was evaluated by additivity and competition assays using antigen-coated plates and by surface plasmon resonance (SPR), an automated biosensor system that is able to retain the conformation integrity of proteins. IPNV-neutralizing MAbs defined a major, conformational-dependent and immunodominant area where continuous epitopes represent portions of a larger discontinuous epitope. Moreover, weakly neutralizing MAbs could interact with internal sites, located within the foldings of the polypeptide chain. Anti-VP2 MAbs prepared against the European serotype N1 recognized and competed for epitopes present on the North American strain LWVRT 60.1 and Jasper. Attempts to establish the proximity of VP2 and VP3 epitopes have been made by SPR. Results indicate that these major structural proteins do not overlap in the virion.

Real-time kinetic studies on the interaction of transforming growth factor alpha with the epidermal growth factor receptor extracellular domain reveal a conformational change model.

Transforming growth factor alpha (TGF-alpha), epidermal growth factor (EGF), and related factors mediate their biological effects by binding to the extracellular domain of the EGF receptor, which leads to activation of the receptor's cytoplasmic tyrosine kinase activity. Much remains to be determined, however, about the detailed molecular mechanism involved in this ligand-induced receptor activation. The determination of the binding mechanism and the related thermodynamic and kinetic parameters are of prime importance. To do so, we have used a surface plasmon resonance-based biosensor (the BIAcore) that allows the real-time recording of the interaction between TGF-alpha and the extracellular domain of the EGF receptor. By immobilizing different biotinylated derivatives of TGF-alpha on the sensor chip surface, we demonstrated that the N-terminus of TGF-alpha is not directly involved in receptor binding. By optimizing experimental conditions and interpreting the biosensor results by several data analysis methods, we were able to show that the data do not fit a simple binding model. Through global analysis of the data using a numerical integration method, we tested several binding mechanisms for the TGF-alpha/EGF receptor interaction and found that a conformational change model best fits the biosensor data. Our results, combined with other analyses, strongly support a receptor activation mechanism in which ligand binding results in a conformation-driven exposure of a dimerization site on the receptor.

The extracellular domain of the epidermal growth factor receptor. Studies on the affinity and stoichiometry of binding, receptor dimerization and a binding-domain mutant.

The binding of epidermal growth factor (EGF) or an EGF-like growth factor to the EGF receptor is the initial event which leads to receptor activation, and consequently the induction of cell growth. In order to study this binding interaction in detail, we produced the extracellular domain of the EGF receptor (EGFR) using the baculovirus expression system. Affinity-labeling and Western-blot analyses revealed that the baculovirus-infected insect cells secrete active EGFR extracellular domain relatively efficiently, however a significant amount of inactive EGFR extracellular domain is retained within the cells. The apparent dissociation constant (Kd) of the secreted EGFR extracellular domain for EGF and transforming growth factor alpha (TGF-alpha), as determined using an immobilized receptor binding assay, was approximately 200 nM. Interestingly, this Kd value is 30-40-fold lower than that of the full-length EGFR derived from detergent-solubilized A431 cell membranes. The stoichiometry of binding of the EGFR extracellular domain to EGF and TGF-alpha was examined by band-shift analysis on non-denaturing PAGE and was estimated to be 1:1. We have also shown, using sedimentation equilibrium analysis, that ligand binding induces significant dimerization of the EGFR extracellular domain. Finally, we carried out site-specific mutagenesis on the EGFR extracellular domain in order to define the ligand-binding region. We identified amino acid residues which are close to the binding site since they are common to the epitopes of several ligand-competitive monoclonal antibodies. However, these residues do not contribute directly to ligand binding since the affinity of the mutated EGFR extracellular domain for EGF and TGF-alpha was unaffected.

Kinetic study on the formation of a de novo designed heterodimeric coiled-coil: use of surface plasmon resonance to monitor the association and dissociation of polypeptide chains.

The surface plasmon resonance (SPR) technique was used to study the formation kinetics of a de novo designed coiled-coil (E/K coil). The E/K coil is made up of two distinct peptides (E and K) each with five heptad (g-a-b-c-d-e-f) repeats. The E peptide's heptad sequence is E-V-S-A-L-E-K, and the K peptide's heptad sequence is K-V-S-A-L-K-E. A linker C-nL-G-G-G (nL = norleucine) is present at the C-terminus of the E peptide and at the N-terminus of the K peptide for the SPR studies. Heterodimer formation involves both electrostatic and hydrophobic interactions at the dimer interface. Under conditions that favor the heterodimer formation, the CD signal ([theta]222) varied as a function of peptide concentration. The estimated dissociation constant (Kd) was 2.45 +/- 0.71 nM. Denaturation studies with guanidine-HCI (GdnHC11/2 = 3.9 M) suggested a value of 3.53 +/- 0.48 nM. For the SPR investigation, the peptides were biotinylated and linked to streptavidin in order to increase their effective molecular weight and consequently enhance the signal intensity. Biotinylation in itself did not impede coiled-coil formation based on CD measurements. The biosensor study revealed a slow dissociation rate constant for the heterodimer (kd approximately 2 x 10(-4) s-1) and a moderately fast association rate constant [ka approximately (4.27-4.53) x 10(5) M-1 s-1). This gives a calculated Kd of 0.47-0.50 nM, which agrees reasonably well with the equilibrium CD studies. Therefore, based on the SPR data, the preference for heterodimer formation is due to a combination of moderately fast association and slow dissociation rates.

Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) are mitogenic hormones that exert their activity primarily by binding to the EGF receptor, also known as ErbB-1. We have recently characterized a set of EGF/TGFalpha chimeric molecules with similar high affinity for ErbB-1 as EGF and TGFalpha and shown that three of these chimeras induce mitogenic cell stimulation at already a 10-fold lower concentration than their wild-type counterparts (Lenferink, A. E., Kramer, R. H., van Vugt, M. J., Königswieser, M., DiFiore, P. P., van Zoelen, E. J., and van de Poll, M. L. (1997) Biochem. J. 327, 859-865). In the present study we show that these so-called superagonistic chimeras do not differ from EGF and TGFalpha in their ability to induce ErbB-1 tyrosine phosphorylation but are considerably more potent in activation of mitogen-activated protein kinase phosphorylation. Direct cell binding studies and analysis of ligand-receptor interaction by surface plasmon resonance measurements revealed that both the association rate constant (k(on)) and the dissociation rate constant (k(off)) of these superagonists is 3-5-fold higher in comparison with the wild-type ligands and nonsuperagonistic chimeras. These data indicate that the dynamic on and off rate constants for receptor binding may be more specific parameters for determining the mitogenic activity of peptide hormones than their constants for equilibrium receptor binding.