Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals

Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity.

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

The new insight into dynamic crossover in glass forming liquids from the apparent enthalpy analysis

One of the most intriguing phenomena in glass forming systems is the dynamic crossover (T(B)), occurring well above the glass temperature (T(g)). So far, it was estimated mainly from the linearized derivative analysis of the primary relaxation time τ(T) or viscosity η(T) experimental data, originally proposed by Stickel et al. [J. Chem. Phys. 104, 2043 (1996); J. Chem. Phys. 107, 1086 (1997)]. However, this formal procedure is based on the general validity of the Vogel-Fulcher-Tammann equation, which has been strongly questioned recently [T. Hecksher et al. Nature Phys. 4, 737 (2008); P. Lunkenheimer et al. Phys. Rev. E 81, 051504 (2010); J. C. Martinez-Garcia et al. J. Chem. Phys. 134, 024512 (2011)]. We present a qualitatively new way to identify the dynamic crossover based on the apparent enthalpy space (H(a)(') = dlnτ/d(1/T)) analysis via a new plot lnH(a)(') vs. 1∕T supported by the Savitzky-Golay filtering procedure for getting an insight into the noise-distorted high order derivatives. It is shown that depending on the ratio between the "virtual" fragility in the high temperature dynamic domain (m(high)) and the "real" fragility at T(g) (the low temperature dynamic domain, m = m(low)) glass formers can be splitted into two groups related to f < 1 and f > 1, (f = m(high)∕m(low)). The link of this phenomenon to the ratio between the apparent enthalpy and activation energy as well as the behavior of the configurational entropy is indicated.

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

Efficacy of telavancin against penicillin-resistant pneumococci and Staphylococcus aureus in a rabbit meningitis model and determination of kinetic parameters

The penetration of telavancin was 2% into inflamed meninges and ca. 1 per thousand into noninflamed meninges after two intravenous injections (30 mg/kg of body weight). In experimental meningitis, telavancin was significantly superior to vancomycin combined with ceftriaxone against a penicillin-resistant pneumococcal strain. Against a methicillin-sensitive staphylococcal strain, telavancin was slightly but not significantly superior to vancomycin.

Apparent diffusion coefficient in the aging mouse brain: a magnetic resonance imaging study

Novel magnetic resonance imaging sequences have and still continue to play an increasing role in neuroimaging and neuroscience. Among these techniques, diffusion-weighted imaging (DWI) has revolutionized the diagnosis and management of diseases such as stroke, neoplastic disease and inflammation. However, the effects of aging on diffusion are yet to be determined. To establish reference values for future experimental mouse studies we tested the hypothesis that absolute apparent diffusion coefficients (ADC) of the normal brain change with age. A total of 41 healthy mice were examined by T2-weighted imaging and DWI. For each animal ADC frequency histograms (i) of the whole brain were calculated on a voxel-by-voxel basis and region-of-interest (ROI) measurements (ii) performed and related to the animals' age. The mean entire brain ADC of mice <3 months was 0.715(+/-0.016) x 10(-3) mm2/s, no significant difference to mice aged 4 to 5 months (0.736(+/-0.040) x 10(-3) mm2/s) or animals older than 9 months 0.736(+/-0.020) x 10(-3) mm2/s. Mean whole brain ADCs showed a trend towards lower values with aging but both methods (i + ii) did not reveal a significant correlation with age. ROI measurements in predefined areas: 0.723(+/-0.057) x 10(-3) mm2/s in the parietal lobe, 0.659(+/-0.037) x 10(-3) mm2/s in the striatum and 0.679(+/-0.056) x 10(-3) mm2/s in the temporal lobe. With advancing age, we observed minimal diffusion changes in the whole mouse brain as well as in three ROIs by determination of ADCs. According to our data ADCs remain nearly constant during the aging process of the brain with a small but statistically non-significant trend towards a decreased diffusion in older animals.

Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.

Noninvasive determination of intraocular pressure (IOP) in nonsedated mice of 5 different inbred strains

BACKGROUND: Noninvasive intraocular pressure (IOP) measurement in mice is critically important for understanding the pathophysiology of glaucoma. Rebound tonometry is one of the methods that can be used for obtaining such measurements. We evaluated the ability of the rebound tonometer (RT) to determine IOP differences among various mouse strains and whether differences in corneal thickness may affect IOP measurements in these animals. MATERIALS AND METHODS: Five different commonly used mouse strains (BALB/C, CBA/CAHN, AKR/J, CBA/J, and 129P3/J) were used. IOP was measured in eyes from 12 nonsedated animals (6 male and 6 female) from each strain at 2 to 3 months of age using the RT. IOPs were measured in all animals, on 2 different days between 10 AM and 12 PM. Subsequently, a number of eyes from each strain were cannulated to provide a calibration curve specific for that strain. Tonometer readings for all strains were converted to apparent IOP values using the calibration data obtained from the calibration curve of the respective strain. For comparison purposes, IOP values were also obtained using the C57BL/6 calibration data previously reported. IOP for the 5 strains, male and female animals, and the different occasion of measurement were compared using repeat measures analysis of variance. The central corneal thickness (CCT) of another group of 8 male animals from each of the 5 strains was also measured using an optical low coherence reflectometry (OLCR) pachymeter modified for use with mice. CCT values were correlated to mean IOPs of male animals and to the slopes and intercept of individual strain calibration curves. RESULTS: Noninvasive IOP measurements confirm that the BALB/C strain has lower and the CBA/CAHN has higher relative IOPs than other mouse strains while the AKR/J, the CBA/J, and the 129P3/J strains have intermediate IOPs. There is a very good correlation of apparent IOP values obtained by RT with previously reported true IOPs obtained by cannulation. There was a small but statistically significant difference in IOP between male and female animals in 2 strains (129P3/J and AKR/J) with female mice having higher relative IOPs. No correlation between CCT and IOP was detected. CCT did not correlate with any of the constants describing the calibration curves in the various strains. CONCLUSIONS: Noninvasive IOP measurement in mice using the RT can be used to help elucidate IOP phenotype, after prior calibration of the tonometer. CCT has no effect on mouse IOP measurements using the RT.

Apparent survival of the salamander

Background Understanding the demographic processes underlying population dynamics is a central theme in ecology. Populations decline if losses from the population (i.e., mortality and emigration) exceed gains (i.e., recruitment and immigration). Amphibians are thought to exhibit little movement even though local populations often fluctuate dramatically and are likely to go exinct if there is no rescue effect through immigration from nearby populations. Terrestrial salamanders are generally portrayed as amphibians with low migratory activity. Our study uses demographic analysis as a key to unravel whether emigration or mortality is the main cause of "losses" from the population. In particular, we use the analysis to challenge the common belief that terrestrial salamanders show low migratory activity. Results The mark-recapture analysis of adult salamanders showed that monthly survival was high (> 90%) without a seasonal pattern. These estimates, however, translate into rather low rates of local annual survival of only ~40% and suggest that emigration was important. The estimated probability of emigration was 49%. Conclusion Our analysis shows that terrestrial salamanders exhibit more migratory activity than commonly thought. This may be due either because the spatial extent of salamander populations is underestimated or because there is a substantial exchange of individuals between populations. Our current results are in line with several other studies that suggest high migratory activity in amphibians. In particular, many amphibian populations may be characterized by high proportions of transients and/or floaters.