A novel estrogen sensor based on recombinant Arxula adeninivorans cells

A novel yeast cell-based assay was developed for the detection of estrogenic activity in wastewater. Recombinant Arxula adeninivorans strains were engineered to co-express the human estrogen receptor alpha (hERalpha) and a Klebsiella-derived phytase (phyK) reporter gene under the control of an A. adeninivorans-derived glucoamylase (GAA) promoter which had been modified by the insertion of estrogen-responsive elements (EREs). In the presence of estrogenic compounds, hERalpha dimerizes and binds to the estrogen. Reporter gene expression is induced by subsequent binding of the hERalpha-dimer/estrogen complex to estrogen responsive elements (ERE) in the promoter. The insertion of different numbers of EREs in three alternative promoter positions and its effect on reporter gene expression were assessed. In one of the constructs, a detection limit of 5 ng l(-1) and a determination limit of 10 ng l(-1) for 17beta-estradiol-like activity was achieved. The photometric assay used enabled estrogen determination in sewage samples within 30 h.     

Calorific and carbon values of marine and freshwater Protozoa

Calorific and carbon values were determined for a variety of marine and freshwater Protozoa (Noctiluca miliaris, Euplotes sp.,Eufolliculina sp. respectivelyTetrahymena pyriformis, Paramecium caudatum), their food sources(Bacteria, Dunaliella primolecta, Ceratium hirundinella), and for Protozoa-dominated plankton samples. Most calorific values lie close to the centre of the range covering organisms in general. Low values in some marine samples probably resulted from the retention of bound water in the dried material. When all results were combined with data selected from the literature, the dependence of calorific value on carbon content was highly significant. This relationship is probably also adequately described by an energy-carbon regression through the variety of organic compounds commonly found in organisms. Calorific value expressed per unit carbon is shown to vary little in Protozoa (mean conversion factor 46 J [mg C]^–1) or throughout the range of biological materials considered in this study (45 J [mg C]^–1).     

Ventilation-induced lung injury and mechanotransduction: stretching it too far?

The Acute Respiratory Distress Syndrome Network clinical trial on ventilation of critically ill patients has drawn attention to the potential side effects of mechanical ventilation. Both clinical and basic research have demonstrated that injurious ventilation strategies can initiate or perpetuate local and systemic inflammatory responses. There are four principal mechanisms that can produce such a response. 1) Ventilation, especially with high ventilation pressures and zero positive end-expiratory pressure, can cause stress failure of the plasma membrane and of epithelial and endothelial barriers. Stress failure of the plasma membrane causes necrosis, which leads to liberation of both preformed inflammatory mediators and agents that stimulate other cells that are still intact to produce such mediators. 2) Stress failure of the barriers causes loss of compartmentalization with spread of mediators and bacteria throughout the body as a consequence. 3) Less injurious ventilation strategies that do not cause tissue destruction can elicit release of mediators by more specific mechanisms, presumably through activation of stretch-activated signaling cascades (mechanotransduction). 4) Ventilation with increasing positive pressures raises the pressure in the pulmonary circulation and thus vascular shear stress, both of which are known stimuli for endothelial cells. These different mechanisms should be taken into account in the design and the interpretation of studies on molecular mechanisms of ventilation-induced lung injury.     

Protein-DNA interaction and CpG methylation at rep*/vIL-10p of latent Epstein-Barr virus genomes in lymphoid cell lines.

The viral interleukin-10 promoter (vIL-10p), overlapping the rep* element in the Epstein-Barr virus (EBV) genome, is a promoter element active mostly in the late phase of the lytic cycle and immediately upon infection of B cells. rep* was, through transfection experiments with small plasmids, characterised as a cis element supporting oriP replicative function. In this study, in vivo protein binding and CpG methylation at rep*/vIL-10p were analysed in five cell lines that harbour strictly latent EBV genomes. Contrary to the invariably unmethylated dyad symmetry element (DS) of oriP, rep*/vIL-10p was highly methylated and showed only traces of protein binding in all examined cell lines. This result is in agreement with vIL-10p being an inactive promoter of EBV genomes, and makes it less likely that rep* functions as a replicative element of latent EBV genomes.     

Pressor responses to platelet-activating factor and thromboxane are mediated by Rho-kinase

Platelet-activating factor (PAF) contracts smooth muscle of airways and vessels primarily via release of thromboxane. Contraction of smooth muscle is thought to be mediated either by calcium and inositol trisphosphate (IP(3))-dependent activation of the myosin light chain kinase or, alternatively, via the recently discovered Rho-kinase pathway. Here we investigated the contribution of these two pathways to PAF and thromboxane receptor-mediated broncho- and vasoconstriction in two different rat models: the isolated perfused lung (IPL) and precision-cut lung slices. Inhibition of the IP(3) receptor (1-10 microM xestospongin C) or inhibition of phosphatidylinositol-specific PLC (30 microM L-108) did not affect bronchoconstriction but attenuated the sustained vasoconstriction by PAF. Inhibition of myosin light chain kinase (35 microM ML-7) or of calmodulin kinase kinase (26 microM STO609), which regulates the phosphorylation of the myosin light chain, had only a small effect on PAF- or thromboxane-induced pressor responses. Similarly, calmidazolium (10 microM), which inhibits calmodulin-dependent proteins, only weakly reduced the airway responses. In contrast, Y-27632 (10 microM), a Rho-kinase inhibitor, attenuated the thromboxane release triggered by PAF and provided partial or complete inhibition against PAF- and thromboxane-induced pressor responses, respectively. Together, our data indicate that PAF- and thus thromboxane receptor-mediated smooth muscle contraction depends largely on the Rho-kinase pathway.     

Arteriovenous carboxyhemoglobin gradient is a technical artifact that is eliminated by special calibration (SAT 100)

Pulmonary enzyme heme oxygenase, which catalyses carbon monoxide production, may be responsible for arteriovenous carboxyhemoglobin (COHb) differences measured in humans. Unspecific inflammatory stimuli have been shown to induce pulmonary heme oxygenase possibly leading to increased pulmonary carbon monoxide production and elevated arterial COHb. Arteriovenous COHb gradients may therefore be a measurable parameter of lung injury severity. To exclude a technical artefact, we repeated measurements of central venous COHb and arterial COHb in healthy humans (ASA I-II) undergoing elective surgery with the ABL 625 and the updated version, ABL 725 (Radiometer, Copenhagen). In addition to the standard calibration, an especially accurate adjustment of the spectrophotometer wavelengths (SAT100) was performed. This adjustment eliminates the FCOHb dependency on the oxygen saturation. No significant differences were detectable between central venous and arterial COHb concentrations with either blood gas analyzer. The difference between central venous COHb and arterial COHb was 0.09 with the ABL 625 and -0.03 with the ABL 725. Therefore, we conclude that previously reported arteriovenous COHb differences are artifactual and may be eliminated by SAT 100 adjustment, as is possible with the ABL 725.     

Bronchoconstriction in nonhuman primates: a species comparison

Bronchoconstriction is a characteristic symptom of various chronic obstructive respiratory diseases such as chronic obstructive pulmonary disease and asthma. Precision-cut lung slices (PCLS) are a suitable ex vivo model to study physiological mechanisms of bronchoconstriction in different species. In the present study, we established an ex vivo model of bronchoconstriction in nonhuman primates (NHPs). PCLS prepared from common marmosets, cynomolgus macaques, rhesus macaques, and anubis baboons were stimulated with increasing concentrations of representative bronchoconstrictors: methacholine, histamine, serotonin, leukotriene D? (LTD?), U46619, and endothelin-1. Alterations in the airway caliber were measured and compared with previously published data from rodents, guinea pigs, and humans. Methacholine induced maximal airway constriction, varying between 74 and 88% in all NHP species, whereas serotonin was ineffective. Histamine induced maximal bronchoconstriction of 77 to 90% in rhesus macaques, cynomolgus macaques, and baboons and a lesser constriction of 53% in marmosets. LTD? was ineffective in marmosets and rhesus macaques but induced a maximum constriction of 44 to 49% in cynomolgus macaques and baboons. U46619 and endothelin-1 caused airway constriction in all NHP species, with maximum constrictions of 65 to 91% and 70 to 81%, respectively. In conclusion, PCLS from NHPs represent a valuable ex vivo model for studying bronchoconstriction. All NHPs respond to mediators relevant to human airway disorders such as methacholine, histamine, U46619, and endothelin-1 and are insensitive to the rodent mast cell product serotonin. Only PCLS from cynomolgus macaques and baboons, however, responded also to leukotrienes, suggesting that among all compared species, these two NHPs resemble the human airway mechanisms best.