Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Liprin (beta)1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity.

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.

Early processing in the human lateral occipital complex is highly responsive to illusory contours but not to salient regions.

Human electrophysiological studies support a model whereby sensitivity to so-called illusory contour stimuli is first seen within the lateral occipital complex. A challenge to this model posits that the lateral occipital complex is a general site for crude region-based segmentation, based on findings of equivalent hemodynamic activations in the lateral occipital complex to illusory contour and so-called salient region stimuli, a stimulus class that lacks the classic bounding contours of illusory contours. Using high-density electrical mapping of visual evoked potentials, we show that early lateral occipital cortex activity is substantially stronger to illusory contour than to salient region stimuli, whereas later lateral occipital complex activity is stronger to salient region than to illusory contour stimuli. Our results suggest that equivalent hemodynamic activity to illusory contour and salient region stimuli probably reflects temporally integrated responses, a result of the poor temporal resolution of hemodynamic imaging. The temporal precision of visual evoked potentials is critical for establishing viable models of completion processes and visual scene analysis. We propose that crude spatial segmentation analyses, which are insensitive to illusory contours, occur first within dorsal visual regions, not the lateral occipital complex, and that initial illusory contour sensitivity is a function of the lateral occipital complex.

Daptomycin for highly resistant Enterococcus faecium infection.

We report a case series of 11 patients with severe E. faecium infections treated with daptomycin. All strains were resistant to ampicillin (MIC >8 mg/l), but susceptible to vancomycin. Seven out of 11 strains were also highly resistant to gentamicin (MIC >500 mg/l). All patients were treated with multiple broad-spectrum antibiotics prior to isolation of E. faecium and had severe underlying diseases. Our experience suggests that salvage therapy with daptomycin might be a safe and efficacious treatment for E. faecium infections.

A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers.

The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented. Copyright © 2011 John Wiley & Sons, Ltd.