Determination of Real-Time Efflux Phenotypes in Escherichia coli AcrB Binding Pocket Phenylalanine Mutants Using a 1,2′-Dinaphthylamine Efflux Assay

To evaluate the importance of phenylalanine residues for substrate transport in the Escherichia coli efflux pump protein AcrB, we subjected Phe-to-Ala binding pocket mutants to a real-time efflux assay with the novel near-infrared lipophilic membrane probe 1,2′-dinaphthylamine (1,2′-DNA). All mutations, with the exception of F617A, led to considerable retardation of efflux. F610A was the point mutation with the most pronounced impact, followed by F628A, F615A, F136A, and F178A. This is the first study to demonstrate the importance of single phenylalanine residues within the AcrB binding pocket for real-time substrate transport

Cognitive factors mediate placebo responses in patients with house dust mite allergy.

BACKGROUND: Placebo effects have been reported in type I allergic reactions. However the neuropsychological mechanisms steering placebo responses in allergies are largely unknown. The study analyzed whether and to what extend a conditioned placebo response is affecting type I allergic reactions and whether this response can be reproduced at multiple occasions. METHODS: 62 patients with house dust mite allergy were randomly allocated to either a conditioned (n = 25), sham-conditioned (n = 25) or natural history (n = 12) group. During the learning phase (acquisition), patients in the conditioned group received the H1-receptor antagonist desloratadine (5mg) (unconditioned stimulus/US) together with a novel tasting gustatory stimulus (conditioned stimulus/CS). Patients in the sham-conditioned control group received the CS together with a placebo pill. After a wash out time of 9 days patients in the conditioned and sham-conditioned group received placebo pills together with the CS during evocation. Allergic responses documented by wheal size after skin prick test and symptom scores after nasal provocation were analyzed at baseline, after last desloratadine treatment and after the 1(st) and 5(th) CS re-exposure. RESULTS: Both conditioned and sham-conditioned patients showed significantly decreased wheal sizes after the 1(st) CS-evocation and significantly decreased symptom scores after the 1(st) as well as after the 5(th) evocation compared to the natural history control group. CONCLUSIONS: These results indicate that placebo responses in type I allergy are not primarily mediated by learning processes, but seemed to be induced by cognitive factors such as patients' expectation, with these effects not restricted to a single evocation

Increased quenching of 1,2′-DNA fluorescence at higher dye loading concentrations (DLCs) in strain 3-AG100.

<p>Depicted are relative fluorescence units (RFU) of deenergized cells preloaded with 1,2′-DNA after resuspension in fresh PPB. All experiments were done in triplicate. The error bars show the standard deviation.</p

Representative 1,2′-DNA efflux curves of 3-AG100-derived AcrB binding pocket mutants.

<p>After preloading with 4 µM 1,2′-DNA the cells were energized at 100 s with 50 mM glucose. Fluorescence intensity is given as relative fluorescence units (RFU) with preenergization levels adjusted to 100 RFU.</p

Psychosocial Stress Increases Salivary Alpha-Amylase Activity Independently from Plasma Noradrenaline Levels.

Salivary alpha-amylase activity (sAA) and plasma noradrenaline (NA) concentrations are often considered to be surrogate markers of sympathetic activation in response to stress. However, despite accumulating evidence for a close association between sAA and noradrenaline and other indicators of sympathetic activity, reliability and generality of this relation remains unclear. We employed the Trier Social Stress Test (TSST) in order to directly compare the responses in sAA and NA to psychological stress in healthy volunteers (n = 23). The TSST significantly increased sAA and NA plasma levels with no significant differences in females and males. However, when subjects were divided according to their NA responses into low versus high responders, both groups did not significantly differ in their sAA before, during or after stress exposure. These data suggest that in response to acute psychological stress both plasma NA levels and sAA reflect sympathetic activity, however seemed to increase independently from each other

Experimental design.

<p>During acquisition, patients in the conditioned group received desloratadine (US) in combination with the CS. During evocation, the drug was replaced by placebo pills. Sham-conditioned patients received the CS together with placebo pills throughout the study. During 3 subsequent days during acquisition (2–4) and evocation (17–19) patients were instructed to intake the pills together with the drink (CS) at home.</p

Sociodemographic and psychological characteristics.

<p>BDI = Becks Depression Inventory; STAI X2 = Trait anxiety form of the State-trait anxiety inventory.</p>*<p>Results of Chi² Test or univariate ANOVA.</p

Conditioned and sham-conditioned patients show reduced allergic reactions during evocation.

<p><b>A</b> After skin prick test wheal size (mm) was analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in the natural history group. <b>B</b> Symptom scores after nasal provocation were analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in natural history group. Data are presented as mean ±SEM. *p<0.05 **p<0.01 ***p<0.001, comparison against natural history group.</p

The distribution of Noradrenaline response to the Trier Social Stress Test among individuals.

<p>The distribution of Noradrenaline response to the Trier Social Stress Test among individuals.</p