Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro

Enantioselective CE with sulfated cyclodextrins as chiral selectors was used to determine the CYP3A4-catalyzed N-demethylation kinetics of ketamine to norketamine and its inhibition in the presence of ketoconazole in vitro. Ketamine, a chiral phencyclidine derivative, was incubated with recombinant human CYP3A4 from a baculovirus expression system as racemic mixture and as single enantiomer. Alkaline liquid/liquid extracts of the samples were analyzed with a pH 2.5 buffer comprising 50 mM Tris and phosphoric acid together with either multiple isomer sulfated β-cyclodextrin (10 mg/mL) or highly sulfated γ-cyclodextrin (2%, w/v). Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively with Michaelis-Menten (incubation of racemic ketamine) and Hill (separate incubation of single enantiomers) kinetics. Data generated in the presence of ketoconazole as the inhibitor could best be fitted to a one-site competitive model and inhibition constants were calculated using the equation of Cheng and Prusoff. No stereoselective difference was observed, but inhibition constants for the incubation of racemic ketamine were found to be larger compared with those obtained with the incubation of single ketamine enantiomers.

The Pararectus approach for anterior intrapelvic management of acetabular fractures: an anatomical study and clinical evaluation

A new anterior intrapelvic approach for the surgical management of displaced acetabular fractures involving predominantly the anterior column and the quadrilateral plate is described. In order to establish five 'windows' for instrumentation, the extraperitoneal space is entered along the lateral border of the rectus abdominis muscle. This is the so-called 'Pararectus' approach. The feasibility of safe dissection and optimal instrumentation of the pelvis was assessed in five cadavers (ten hemipelves) before implementation in a series of 20 patients with a mean age of 59 years (17 to 90), of whom 17 were male. The clinical evaluation was undertaken between December 2009 and December 2010. The quality of reduction was assessed with post-operative CT scans and the occurrence of intra-operative complications was noted. In cadavers, sufficient extraperitoneal access and safe instrumentation of the pelvis were accomplished. In the patients, there was a statistically significant improvement in the reduction of the fracture (pre- versus post-operative: mean step-off 3.3 mm (sd 2.6) vs 0.1 mm (sd 0.3), p < 0.001; and mean gap 11.5 mm (sd 6.5) vs 0.8 mm (sd 1.3), p < 0.001). Lesions to the peritoneum were noted in two patients and minor vascular damage was noted in a further two patients. Multi-directional screw placement and various plate configurations were feasible in cadavers without significant retraction of soft tissues. In the treatment of acetabular fractures predominantly involving the anterior column and the quadrilateral plate, the Pararectus approach allowed anatomical restoration with minimal morbidity related to the surgical access.

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.

Modulation of human osteoblasts by metal surface chemistry

The use of metal implants in dental and orthopedic surgery is continuously expanding and highly successful. While today longevity and load-bearing capacity of the implants fulfill the expectations of the patients, acceleration of osseointegration would be of particular benefit to shorten the period of convalescence. To further clarify the options to accelerate the kinetics of osseointegration, within this study, the osteogenic properties of structurally identical surfaces with different metal coatings were investigated. To assess the development and function of primary human osteoblasts on metal surfaces, cell viability, differentiation, and gene expression were determined. Titanium surfaces were used as positive, and surfaces coated with gold were used as negative controls. Little differences in the cellular parameters tested for were found when the cells were grown on titanium discs sputter coated with titanium, zirconium, niobium, tantalum, gold, and chromium. Cell number, activity of cell layer-associated alkaline phosphatase (ALP), and levels of transcripts encoding COL1A1 and BGLAP did not vary significantly in dependence of the surface chemistry. Treatment of the cell cultures with 1,25(OH)2 D3 /Dex, however, significantly increased ALP activity and BGLAP messenger RNA levels. The data demonstrate that the metal layer coated onto the titanium discs exerted little modulatory effects on cell behavior. It is suggested that the microenvironment regulated by the peri-implant tissues is more effective in regulating the tissue response than is the material of the implant itself.

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

The Effect Of Osteoprotection On Immune Reconstitution Post Bone Marrow Transplant

Specialized microenvironments have been known to strongly influence stem cell fate in hematopoiesis. The interplay between osteolineage cells, specifically the mature osteoblast, and the hematopoietic stem cell (HSC) niche have been of particular note. Recently, preliminary unpublished data obtained in the Scadden laboratory suggests the critical role of the osteoblast in regulating T cells. The goal of this project was to initially determine whether stimulating the osteoblast in the HSC niche leads to increased immune reconstitution after hematopoietic stem cell transplant (HSCT). These results indicated that while bone manipulation pre-transplant may have a positive effect on T and B lymphocyte cell recovery, bone manipulation post-transplant seems to have a suppressing effect. Additionally, stimulation of the osteoblast may have an inhibitory effect on the regeneration of GR1+ myeloid cells. Based on these results, we then sought to determine how osteoprotection pre-HSCT modifies the kinetics of graft-versus-host disease (GVHD) and impacts the regeneration of immune cells. The data from this phase of my experiment suggests a possible immediate benefit in stimulation of the osteoblast in response to GVHD prior to HSCT. The overall results from my thesis project demonstrate a promising relationship between pre-HSCT stimulation of the osteoblast and lymphocyte recovery post-HSCT. ¿

Evaluation of smell and taste in patients with Wegener's granulomatosis

Although a reduced olfactory/gustatory function affects patients in all parts of life, this problem has not received much attention in Wegener's granulomatosis (WG). The aim of this study was to assess the smell/taste function of WG patients. Demographic data of 16 WG patients (9 males, 7 females) were obtained. They all subjectively assessed their taste/smell function on visual analogue scale. Olfactory/gustatory functions of the patients were tested with 'Sniffin' Sticks and 'Taste' strips, respectively. The results were then compared with those from sex and age-matched control group (n = 16) and normative data. WG patients subjectively assessed their olfactory (p = 0.03) and gustatory (p = 0.02) function to be lower than control group. All the olfactory scores (odour identification, odour discrimination and threshold) in both genders were significantly below the scores in the control group. WG patients were hyposmic. For taste (total taste score, as well as scores for the qualities sweet, sour, salty and bitter), WG patients did not significantly differ from controls and were normogeusic. However, the gustatory scores showed the tendency of reduction as compared to the control group. In conclusion, WG patients truly suffer from olfactory/taste dysfunction, but this is worse with olfaction. It is, therefore, imperative that physicians should make their patients to be aware of these sensory dysfunctions and educate them on methods to cope with it for better quality of life.

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

14-3-3- and II/3-10-gene expression as molecular markers to address viability and growth activity of Echinococcus multilocularis metacestodes

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

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.

Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

Infliximab inhibits bone resorption by circulating osteoclast precursor cells in patients with Rheumatoid Arthritis and Ankylosing Spondylitis

OBJECTIVE: To examine the effects of infliximab on bone resorption by osteoclast precursor cells (OCPs) in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS) and to compare the results with changes in disease activity. METHODS: Before and during 24 weeks of infliximab treatment peripheral blood mononuclear cells of 9 RA and 10 AS patients were seeded onto ivory wafers and adherent cells, including OCPs, were grown in medium promoting osteoclast differentiation. Bone resorption was evaluated morphometrically and correlated to disease activity. 19 healthy individuals were studied in parallel. In addition, biochemical bone markers were assessed in all patients at baseline and after 24 weeks. RESULTS: OCPs from RA patients showed a higher bone resorption at baseline when compared to AS patients. Blocking of TNFalpha with infliximab resulted in a strong reduction of bone resorption by OCPs in both cohorts and did occur faster in RA compared to AS patients. This inhibition coincided with reduction of clinical disease activity in both patient cohorts and with an increase of serum osteocalcin levels and a relative decrease of collagen crosslinks in RA compared to AS patients. CONCLUSION: These results provide an explanation on the cellular level for the anticatabolic effect of TNF neutralization on bone. The variation in the kinetics of bone resorption by the OCPs in patients with RA and AS suggests disease-specific differences in the type or in the preactivation of OCPs.

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Study of the effect of biodiesel fuel on passive oxidation in a catalyzed particulate filter

A 2007 Cummins ISL 8.9L direct-injection common rail diesel engine rated at 272 kW (365 hp) and 317 kW (425 hp) was used to load the filter to 2.2 g/L and passively oxidize particulate matter (PM) within an aftertreatment system consisting of a diesel oxidation catalyst (DOC) and catalyzed particulate filter (CPF). The tests conducted with the engine rated at 365 hp used a 2007 DOC and CPF. The tests conducted with the engine rated at 425 hp used a 2010 DOC and 2007 CPF. Understanding the passive NO2 oxidation kinetics of PM within the CPF allows for reducing the frequency of active regenerations (hydrocarbon injection) and the associated fuel penalties. Modeling the passive oxidation of accumulated PM in the CPF will lead to creating accurate state estimation strategies. The MTU 1-D CPF model will be used to simulate data collected from this study to examine differences in the PM oxidation kinetics when soy methyl ester (SME) biodiesel is used as the source of fuel for the engine, and when the engine is operated at a higher power rating. A test procedure developed by Hutton et al. [1, 2] was modified to improve the ability to model the experimental data and provide additional insight into passively oxidized PM in a partially regenerated CPF. A test procedure was developed to allow PM oxidation rates by NO2 to be determined from engine test cell data. An experimental matrix consisting of CPF inlet temperatures from 250 to 450 °C with varying NOX/PM from 25 to 583and NO2/PM ratios from 5 to 240 was used. SME biodiesel was volumetrically blended with ULSD in 10% (B10) and 20% (B20) portions. This blended fuel was then used to evaluate the effect of biodiesel on passive oxidation rates. Four tests were performed with B10 and four tests with B20. Gathering data to determine the effect of fuel type (ULSD and biodiesel blends) on PM oxidation is the primary goal. The engine used for this testing was then configured to a higher power rating and one of the tests planned was performed. Additional testing is scheduled to take place with ULSD fuel to determine the affect the engine rating has on the PM oxidation. The experimental reaction rates during passive oxidation varied based upon the average CPF temperature, NO2 concentrations, and the NOX/PM ratios for each engine rating and with all fuels. The data analysis requires a high fidelity model that includes NO2 and thermal oxidation mechanisms and back diffusion to determine the details of the PM oxidation process.

Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration

BACKGROUND & AIMS: Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. METHODS: Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. RESULTS: After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. CONCLUSIONS: Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.