Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

Ground reaction force profiles after partial and pancarpal arthrodesis in dogs

OBJECTIVES: To evaluate and compare long-term functional outcome after partial carpal arthrodesis and pancarpal arthrodesis in dogs using kinetic gait analysis. METHODS: Fourteen dogs with 19 partial carpal or pancarpal arthrodeses were retrospectively examined and underwent force-plate gait analysis. Mean times since surgery were 29.4 and 24.4 months for pancarpal and partial carpal arthrodesis respectively. Vertical and braking-propulsive ground reaction force profiles were compared between treatment groups, and to those of normal dogs (control group) using Kruskal-Wallis one-way analysis of variance. RESULTS: With the exception of time to vertical peak that occurred earlier in dogs with pancarpal than in dogs with partial carpal arthrodesis (p <0.01), there was no difference between the two treatment groups. Several parameters differed significantly between operated and healthy dogs (p <0.01): vertical impulses were significantly lower in both treatment groups, braking forces and impulses were also reduced after both techniques. Propulsive forces and impulses were only reduced in dogs with pancarpal arthrodesis. When comparing gait parameters of sound limbs of unilateral operated dogs to those of control dogs, braking forces and impulses (p <0.01; p <0.05) were significantly higher in the sound legs of unilateral operated dogs. CLINICAL SIGNIFICANCE: Long-term outcome after partial carpal and pancarpal arthrodesis is good and comparable to each other. Propulsive action may be altered more in dogs with pancarpal arthrodesis.

Understanding Electrophoretic Stacking Of In-Capillary Generated Reaction Products

The separation of small molecules by capillary electrophoresis is governed by a complex interplay among several physical effects. Until recently, a systematic understanding of how the influence of all of these effects is observed experimentally has remained unclear. The work presented in this thesis involves the use of transient isotachophoretic stacking (tITP) and computer simulation to improve and better understand an in-capillary chemical assay for creatinine. This assay involves the use of electrophoretically mediated micro-analysis (EMMA) to carry out the Jaffé reaction inside a capillary tube. The primary contribution of this work is the elucidation of the role of the length and concentration of the hydroxide plug used to achieve tITP stacking of the product formed by the in-capillary EMMA/Jaffé method. Computer simulation using SIMUL 5.0 predicts that a 3-4 fold gain in sensitivity can be recognized by timing the tITP stacking event such that the Jaffé product peak is at its maximum height as that peak is electrophoresing past the detection window. Overall, the length of the hydroxide plug alters the timing of the stacking event and lower concentration plugs of hydroxide lead to more rapidly occurring tITP stacking events. Also, the inclusion of intentional tITP stacking in the EMMA/Jaffé method improves the sensitivity of the assay, including creatinine concentrations within the normal biological range. Ultimately, improvement in assay sensitivity can be rationally designed by using the length and concentration of the hydroxide plug to engineer the timing of the tITP stacking event such that stacking occurs as the Jaffé product is passing the detection window.

Studying Electrophoretic Separations Using Cholate Micelles: Effects Of Ph, Temperature, And Composition

Micelle-forming bile salts have previously been shown to be effective pseudo-stationary phases for separating the chiral isomers of binaphthyl compounds with micellar electrokinetic capillary chromatography (MEKC). Here, cholate micelles are systematically investigated via electrophoretic separations and NMR using R, S-1, 1¿- binaphthyl- 2, 2¿-diylhydrogenphosphate (BNDHP) as a model chiral analyte. The pH, temperature, and concentration of BNDHP were systematically varied while monitoring the chiral resolution obtained with MEKC and the chemical shift of various protons in NMR. NMR data for each proton on BNDHP is monitored as a function of cholate concentration: as cholate monomers begin to aggregate and the analyte molecules begin to sample the micelle aggregate we observe changes in the cholate methyl and S-BNDHP proton chemical shifts. From such NMR data, the apparent CMC of cholate at pH 12 is found to be about 13-14 mM, but this value decreases at higher pH, suggesting that more extreme pHs may give rise to more effective separations. In general, CMCs increase with temperature indicating that one may be able to obtain better separations at lower temperatures. S-BNDHP concentrations ranging from 50 ¿M to 400 ¿M (pH 12.8) gave rise to apparent cholate CMC values from 10 mM to 8 mM, respectively, indicating that S-BNDHP, the chiral analyte molecule, may play an active role in stabilizing cholate aggregates. In all, these data show that NMR can be used to systematically investigate a complex multi-variable landscape of potential optimizations of chiral separations.

Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.

Validation of MIPAS IMK/IAA temperature, water vapor, and ozone profiles with MOHAVE-2009 campaign measurements

MIPAS observations of temperature, water vapor, and ozone in October 2009 as derived with the scientific level-2 processor run by Karlsruhe Institute of Technology (KIT), Institute for Meteorology and Climate Research (IMK) and CSIC, Instituto de Astrofísica de Andalucía (IAA) and retrieved from version 4.67 level-1b data have been compared to co-located field campaign observations obtained during the MOHAVE-2009 campaign at the Table Mountain Facility near Pasadena, California in October 2009. The MIPAS measurements were validated regarding any potential biases of the profiles, and with respect to their precision estimates. The MOHAVE-2009 measurement campaign provided measurements of atmospheric profiles of temperature, water vapor/relative humidity, and ozone from the ground to the mesosphere by a suite of instruments including radiosondes, ozonesondes, frost point hygrometers, lidars, microwave radiometers and Fourier transform infra-red (FTIR) spectrometers. For MIPAS temperatures (version V4O_T_204), no significant bias was detected in the middle stratosphere; between 22 km and the tropopause MIPAS temperatures were found to be biased low by up to 2 K, while below the tropopause, they were found to be too high by the same amount. These findings confirm earlier comparisons of MIPAS temperatures to ECMWF data which revealed similar differences. Above 12 km up to 45 km, MIPAS water vapor (version V4O_H2O_203) is well within 10% of the data of all correlative instruments. The well-known dry bias of MIPAS water vapor above 50 km due to neglect of non-LTE effects in the current retrievals has been confirmed. Some instruments indicate that MIPAS water vapor might be biased high by 20 to 40% around 10 km (or 5 km below the tropopause), but a consistent picture from all comparisons could not be derived. MIPAS ozone (version V4O_O3_202) has a high bias of up to +0.9 ppmv around 37 km which is due to a non-identified continuum like radiance contribution. No further significant biases have been detected. Cross-comparison to co-located observations of other satellite instruments (Aura/MLS, ACE-FTS, AIRS) is provided as well.

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

High-resolution computer simulation of the dynamics of isoelectric focusing using carrier ampholytes: focusing with concurrent electrophoretic mobilization is an isotachophoretic process

Focusing of four hemoglobins with concurrent electrophoretic mobilization was studied by computer simulation. A dynamic electrophoresis simulator was first used to provide a detailed description of focusing in a 100-carrier component, pH 6-8 gradient using phosphoric acid as anolyte and NaOH as catholyte. These results are compared to an identical simulation except that the catholyte contained both NaOH and NaCl. A stationary, steady-state distribution of carrier components and hemoglobins is produced in the first configuration. In the second, the chloride ion migrates into and through the separation space. It is shown that even under these conditions of chloride ion flux a pH gradient forms. All amphoteric species acquire a slight positive charge upon focusing and the whole pattern is mobilized towards the cathode. The cathodic gradient end is stable whereas the anodic end is gradually degrading due to the continuous accumulation of chloride. The data illustrate that the mobilization is a cationic isotachophoretic process with the sodium ion being the leading cation. The peak height of the hemoglobin zones decreases somewhat upon mobilization, but the zones retain a relatively sharp profile, thus facilitating detection. The electropherograms that would be produced by whole column imaging and by a single detector placed at different locations along the focusing column are presented and show that focusing can be commenced with NaCl present in the catholyte at the beginning of the experiment. However, this may require detector placement on the cathodic side of the catholyte/sample mixture interface.

GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about the effects of GFLs on other neuronal populations in the VM is essential for their potential application as therapeutic molecules for Parkinson's disease. Hence, in a comparative study, we investigated the effects of GFLs on cell densities and morphological differentiation of gamma-aminobutyric acid-immunoreactive (GABA-ir) and serotonin-ir (5-HT-ir) neurons in primary cultures of E14 rat VM. We observed that all GFLs [10 ng/ml] significantly increased GABA-ir cell densities (1.6-fold) as well as neurite length/neuron. However, only GDNF significantly increased the number of primary neurites/neuron, and none of the GFLs affected soma size of GABA-ir neurons. In contrast, only NRTN treatment significantly increased 5-HT-ir cells densities at 10 ng/ml (1.3-fold), while an augmentation was seen for GDNF and PSPN at 100 ng/ml (2.4-fold and 1.7-fold, respectively). ARTN had no effect on 5-HT-ir cell densities. Morphological analysis of 5-HT-ir neurons revealed a significant increase of soma size, number of primary neurites/neuron and neurite length/neuron after GDNF exposure, while PSPN only affected soma size, and NRTN and ARTN failed to exert any effect. In conclusion, we identified GFLs as effective neurotrophic factors for VM GABAergic and serotonergic neurons, demonstrating characteristic individual action profiles emphasizing their important and distinct roles during brain development.