Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta(2)-adrenergic receptor.

1. We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human beta(2) adrenergic receptor (beta(2)AR). 2. A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type beta(2)AR. 3. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4. We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the beta-ethanolamine tail with other residues of the beta(2)-AR binding pocket.

A simple gas chromatography method for the analysis of monoethanolamine in air

A simple method determining airborne monoethanolamine has been developed. Monoethanolamine determination has traditionally been difficult due to analytical separation problems. Even in recent sophisticated methods, this difficulty remains as the major issue often resulting in time-consuming sample preparations. Impregnated glass fiber filters were used for sampling. Desorption of monoethanolamine was followed by capillary GC analysis and nitrogen phosphorous selective detection. Separation was achieved using a specific column for monoethanolamines (35% diphenyl and 65% dimethyl polysiloxane). The internal standard was quinoline. Derivatization steps were not needed. The calibration range was 0.5-80 μg/mL with a good correlation (R(2) = 0.996). Averaged overall precisions and accuracies were 4.8% and -7.8% for intraday (n = 30), and 10.5% and -5.9% for interday (n = 72). Mean recovery from spiked filters was 92.8% for the intraday variation, and 94.1% for the interday variation. Monoethanolamine on stored spiked filters was stable for at least 4 weeks at 5°C. This newly developed method was used among professional cleaners and air concentrations (n = 4) were 0.42 and 0.17 mg/m(3) for personal and 0.23 and 0.43 mg/m(3) for stationary measurements. The monoethanolamine air concentration method described here was simple, sensitive, and convenient both in terms of sampling and analytical analysis.

Airborne exposures to monoethanolamine, glycol ethers, and benzyl alcohol during professional cleaning: a pilot study

A growing body of epidemiologic evidence suggests an association between exposure to cleaning products and respiratory dysfunction. Due to the lack of quantitative assessments of respiratory exposures to airborne irritants and sensitizers among professional cleaners, the culpable substances have yet to be identified.Purpose: Focusing on previously identified irritants, our aims were to determine (i) airborne concentrations of monoethanolamine (MEA), glycol ethers, and benzyl alcohol (BA) during different cleaning tasks performed by professional cleaning workers and assess their determinants; and (ii) air concentrations of formaldehyde, a known indoor air contaminant. METHODS: Personal air samples were collected in 12 cleaning companies, and analyzed by conventional methods. RESULTS: Nearly all air concentrations [MEA (n = 68), glycol ethers (n = 79), BA (n = 15), and formaldehyde (n = 45)] were far below (<1/10) of the corresponding Swiss occupational exposure limits (OEL), except for ethylene glycol mono-n-butyl ether (EGBE). For butoxypropanol and BA, no OELs exist. Although only detected once, EGBE air concentrations (n = 4) were high (49.48-58.72mg m(-3)), and close to the Swiss OEL (49mg m(-3)). When substances were not noted as present in safety data sheets of cleaning products used but were measured, air concentrations showed no presence of MEA, while the glycol ethers were often present, and formaldehyde was universally detected. Exposure to MEA was affected by its amount used (P = 0.036), and spraying (P = 0.000) and exposure to butoxypropanol was affected by spraying (P = 0.007) and cross-ventilation (P = 0.000). CONCLUSIONS: Professional cleaners were found to be exposed to multiple airborne irritants at low concentrations, thus these substances should be considered in investigations of respiratory dysfunctions in the cleaning industry; especially in specialized cleaning tasks such as intensive floor cleaning.

Enhanced-reality video fluorescence: a real-time assessment of intestinal viability.

OBJECTIVE: Our aim was to evaluate a fluorescence-based enhanced-reality system to assess intestinal viability in a laparoscopic mesenteric ischemia model. MATERIALS AND METHODS: A small bowel loop was exposed, and 3 to 4 mesenteric vessels were clipped in 6 pigs. Indocyanine green (ICG) was administered intravenously 15 minutes later. The bowel was illuminated with an incoherent light source laparoscope (D-light-P, KarlStorz). The ICG fluorescence signal was analyzed with Ad Hoc imaging software (VR-RENDER), which provides a digital perfusion cartography that was superimposed to the intraoperative laparoscopic image [augmented reality (AR) synthesis]. Five regions of interest (ROIs) were marked under AR guidance (1, 2a-2b, 3a-3b corresponding to the ischemic, marginal, and vascularized zones, respectively). One hour later, capillary blood samples were obtained by puncturing the bowel serosa at the identified ROIs and lactates were measured using the EDGE analyzer. A surgical biopsy of each intestinal ROI was sent for mitochondrial respiratory rate assessment and for metabolites quantification. RESULTS: Mean capillary lactate levels were 3.98 (SD = 1.91) versus 1.05 (SD = 0.46) versus 0.74 (SD = 0.34) mmol/L at ROI 1 versus 2a-2b (P = 0.0001) versus 3a-3b (P = 0.0001), respectively. Mean maximal mitochondrial respiratory rate was 104.4 (±21.58) pmolO2/second/mg at the ROI 1 versus 191.1 ± 14.48 (2b, P = 0.03) versus 180.4 ± 16.71 (3a, P = 0.02) versus 199.2 ± 25.21 (3b, P = 0.02). Alanine, choline, ethanolamine, glucose, lactate, myoinositol, phosphocholine, sylloinositol, and valine showed statistically significant different concentrations between ischemic and nonischemic segments. CONCLUSIONS: Fluorescence-based AR may effectively detect the boundary between the ischemic and the vascularized zones in this experimental model.

Evaluation of respiratory and dermal occupational exposures to monoethanolamine during cleaning

Professional cleaning is a basic service occupation with a wide variety of tasks carried out in all kind of different sectors and workplaces by a large workforce. One important risk for cleaning workers is the exposure to chemical substances that are present in cleaning products.Monoethanolamine was found to be often present in cleaning products such as general purpose cleaners, bathroom cleaners, floor cleaners and kitchen cleaners. Monoethanolamine can injure the skin, and exposure to monoethanolamine was associated to asthma even when the air concentrations were low. It is a strong irritant and known to be involved in sensitizing mechanisms. It is very likely that the use of cleaning products containing monoethanolamine gives rise to respiratory and dermal exposures. Therefore there is a need to further investigate the exposures to monoethanolamine for both, respiratory and dermal exposure.The determination of monoethanolamine has traditionally been difficult and analytical methods available are little adapted for occupational exposure assessments. For monoethanolamine air concentrations, a sampling and analytical method was already available and could be used. However, a method to analyses samples for skin exposure assessments as well as samples of skin permeation experiments was missing. Therefore one main objective of this master thesis was to search an already developed and described analytical method for the measurement of monoethanolamine in water solutions, and to set it up in the laboratory. Monoethanolamine was analyzed after a derivatisation reaction with o-pthtaldialdehyde. The derivated fluorescing monoethanolamine was then separated with high performance liquid chromatography and detection took place with a fluorescent detector. The method was found to be suitable for qualitative and quantitative analysis of monoethanolamine. An exposure assessment was conducted in the cleaning sector to measure the respiratory and dermal exposures to monoethanolamine during floor cleaning. Stationary air samples (n=36) were collected in 8 companies and samples for dermal exposures (n=12) were collected in two companies. Air concentrations (Mean = 0.18 mg/m3, Standard Deviation = 0.23 mg/m3, geometric Mean = 0.09 mg/m3, Geometric Standard Deviation = 3.50) detected were mostly below 1/10 of the Swiss 8h time weighted average occupational exposure limit. Factors that influenced the measured monoethanolamine air concentrations were room size, ventilation system and the concentration of monoethanolamine in the cleaning product and amount of monoethanolamine used. Measured skin exposures ranged from 0.6 to 128.4 mg/sample. Some cleaning workers that participated in the skin exposure assessment did not use gloves and had direct contact with the solutions containing the cleaning product and monoethanolamine. During the entire sampling campaign, cleaning workers mostly did not use gloves. Cleaning workers are at risk to be regularly exposed to low air concentrations of monoethanolamine. This exposure may be problematic if a worker suffers from allergic reactions (e.g. Asthma). In that case a substitution of the cleaning product may be a good prevention measure as several different cleaning products are available for similar cleaning tasks. Currently there are no occupational exposure limits to compare the skin exposures that were found. To prevent skin exposures, adaptations of the cleaning techniques and the use of gloves should be considered. The simultaneous skin and airborne exposures might accelerate adverse health effects. Overall the risks caused by exposures to monoethanolamine are considered as low to moderate when the cleaning products are used correctly. Whenever possible, skin exposures should be avoided. Further research should consider especially the dermal exposure routes, as very high exposures might occur by skin contact with cleaning products. Dermatitis but also sensitization might be caused by skin exposures. In addition, new biomedical insights are needed to better understand the risks of the dermal exposure. Therefore skin permeability experiments should be considered.

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.