Kinetics of chemically mediated neurodegeneration/neuroregeneration of mouse olfactory epithelium: monitoring by hyperlayer sedimentation field flow fractionation.

The increase in the incidence of neurodegenerative diseases linked to aging or injury needs to be addressed in research into neuroprotective or neuroregenerative therapies, and requires the development of specific biological models. To achieve this goal we propose (1) the use of the mouse olfactory epithelium as a biological support which specifically exhibits a regenerative or a self-renewing capacity and during the lifetime necessitates the presence of neural stem cells, and (2) the use of an intraperitoneal injection of 2,6-dichlorobenzonitrile (diclobenil) as a chemical inducer of neurodegeneration in olfactory epithelium by selectively killing mature cells. We developed a biological model to follow the processes of neurodegeneration (chemically induced) and neuroregeneration (self-renewal of olfactory epithelium). The purpose of this study was to develop a method to monitor quickly neurodegeneration/neuroregeneration processes in order to further screen protective and regenerative therapies. For this purpose, we used the sedimentation field flow fractionation elution of olfactory epithelium. We obtained specific elution profiles and retention parameters allowing the monitoring of the induction and kinetics of biological processes. The use of insulin-like growth factor 1α as a neuroprotective agent in an innovative nebulization protocol showed sedimentation field flow fractionation to be a simple, fast and low-cost method to monitor such a biological event on the scale of an entire organism.

Measurement of the positive muon lifetime and determination of the Fermi constant to part-per-million precision.

We report a measurement of the positive muon lifetime to a precision of 1.0 ppm; it is the most precise particle lifetime ever measured. The experiment used a time-structured, low-energy muon beam and a segmented plastic scintillator array to record more than 2×10(12) decays. Two different stopping target configurations were employed in independent data-taking periods. The combined results give τ(µ(+)) (MuLan)=2 196 980.3(2.2) ps, more than 15 times as precise as any previous experiment. The muon lifetime gives the most precise value for the Fermi constant: G(F) (MuLan)=1.166 378 8(7)×10(-5) GeV(-2) (0.6 ppm). It is also used to extract the µ(-)p singlet capture rate, which determines the proton's weak induced pseudoscalar coupling g(P).

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

Study of diosgenin-induced apoptosis kinetics in K562 cells by Sedimentation Field Flow Fractionation.

Recently, the use of SdFFF in cancer research has been studied in order to better understand major phenomena implicated in cancer development and therapy: apoptosis and differentiation. In this report, we used SdFFF as a monitoring and cell separation tool to study the kinetics of apoptosis. Incubation of K562 cells with diosgenin, used as cellular model, led to a surprising apoptotic process occurring in two phases (after 24 and 48 h incubation), associated with specific p-ERK expression. Based on the capacity to sort apoptotic cells, results showed that SdFFF cell separation was an effective analytical tool to obtain different subpopulations regardless of the kinetics and extent of apoptosis. Results also showed that, after proper biological calibration of elution profiles, SdFFF can be used to monitor either the induction or the kinetics of a biological event.

Study of the phenotypic relationship in the IMR-32 human neuroblastoma cell line by sedimentation field flow fractionation.

Neuroblastoma (NB) is the most common childhood solid tumor. Although spontaneous regression can occur in patients <1-year old, 70% of patients over the age of 1 year have a high-risk and difficult-to-treat NB. Cell type heterogeneity is observed either in the morphological appearance of NB tumors or in cell lines isolated from tumor specimens. NB consists of two principal neoplastic cell types: i) neuroblastic or N-type (undifferentiated cells); and ii) stromal or S-type (differentiated cells). As NB cells seem to have the capacity to differentiate spontaneously in vivo and in vitro, their heterogeneity could affect treatment outcome, in particular the response to apoptosis induced by chemotherapy. Therefore, it is important to understand the underlying process governing changes in differentiation in order to improve treatment response and NB patient outcome and the neoplastic population in IMR-32 represented a good model for such a study. Results showed that this cell line was extremely heterogeneous and highly variable in its stage of differentiation and we demonstrated that sedimentation field flow fractionation (SdFFF) permitted the isolation of 2 N-phenotypes and contributed to the understanding of the IMR-32 cell population dynamics. The first N-phenotype forms a pool of quiescent undifferentiated cells while the second one was able to proliferate (incorporation of BrdU) and also give rise to adherent S-type cells (PSA-N-CAM+ and N-CAM+). The results could also suggest a close interaction between these different cellular phenotypes to create the IMR-32 cell lineage.

Sedimentation field flow fractionation to study human erythroleukemia cell megakaryocytic differentiation after short period diosgenin induction.

Anti-cancer differentiation therapy could be one strategy to stop cancer cell proliferation. We propose a new sedimentation field flow fractionation (SdFFF) cell separation application in the field of cancer research. It concerns the study of megakaryocytic differentiation processes after a short exposure to an inducting agent (diosgenin). Washout process and early dual SdFFF separation--removing the influence of diosgenin and decreasing the influence of undifferentiated cells--resulted in the preparation of an enriched population to study the mechanism and kinetics of megakaryocytic differentiation. A short exposure to diosgenin was able to induce complete differentiation leading to maximal maturation which ended naturally after 192h incubation without the influence of a secondary effect of diosgenin. The study of isolated undifferentiated cells also showed that no resistance to diosgenin was observed. This result suggested different sensitivities to differentiation induction, and SdFFF cell separation would be of great interest to explore this phenomena.

Sedimentation field flow fractionation monitoring of bimodal wheat starch amylolysis.

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this study, we investigated the capacity of sedimentation field flow fractionation (SdFFF) to monitor the amylolysis of a bimodal starch population: native wheat starch. Results demonstrated a correlation between fractogram changes and enzymatic hydrolysis. Furthermore, SdFFF was used to sort sub-populations which enhanced the study of granule size distribution changes occurring during amylolysis. These results show the interest in coupling SdFFF with particle size measurement methods to study complex starch size/density modifications associated to hydrolysis. These results suggested different applications such as the association of SdFFF with structural investigations to better understand the specific mechanisms of amylolysis or starch granule structure.

Pre-apoptotic sub-population cell sorting from diosgenin apoptosis induced 1547 cells by Sedimentation Field-Flow Fractionation. The effect of channel thickness on sorting performance.

Apoptosis is one of the most important phenomena in cell biology. Pre-apoptotic cells, defined as cells engaged in early stages of apoptosis, could be used as a cellular tool to study apoptosis pathways. The human 1547 osteosarcoma cell line and diosgenin (a plant steroid) association was selected as an in vitro cellular apoptosis model. In a previous study, using this model, we demonstrated that SdFFF monitored apoptosis induction as early as 6h after incubation. In this study, we investigated the capacity of Sedimentation Field-Flow Fractionation (SdFFF) to sort an enriched population of pre-apoptotic cells from 1547 cells incubated for 6 h with 40 microM diosgenin. In that way, two different separation devices which differed especially in channel thickness, 125 and 175 microm, were used and compared. Results showed, for the first time, that SdFFF is an effective method to obtain an enriched pre-apoptotic sub-population. These results suggest, as a new application, that SdFFF could be an included tool in the study of apoptotic mechanisms or the kinetic action of apoptotic drugs.

Neural stem cell separation from the embryonic avian olfactory epithelium by sedimentation field-flow fractionation.

The aim of the present study was to isolate neural stem cells from a complex tissue: the avian olfactory epithelium; by using sedimentation field flow fractionation (SdFFF). By using "Hyperlayer" elution mode, fraction collection and cell characterization methods, results shows that SdFFF could be a useful cell sorter to isolate an enriched, viable and sterile immature neural cell fraction from which the reconstitution of a complete epithelium was possible. In culture, SdFFF eluted cells first led to a "pseudoplacodal" epithelioid cell type from which derived "floating cells". These cells were then able to generate neurosphere-like structures which were composed of cell having many features of immature cells: undifferentiated, self-renewable and multipotentiality. Such a population might be used as a model to improve our understanding of the mechanisms of olfactory neoneurogenesis.

Stabilization of potato tuber lipoxygenase on talc.

Potato tuber lipoxygenase, like many plant enzymes is a very labile protein and can lose activity during or after the purification procedure. In order to overcome this problem, we immobilized potato tuber lipoxygenase by adsorption on talc, a silicate support. Lipoxygenase adsorption greatly increased the long-term stability of the enzyme without modifying enzyme specificity. Moreover, adsorption on talc could be used to purify labile enzymes whose enzymatic activity decreases rapidly during purification stages. A multi-step reaction to produce large quantities (around 100 mg) of fatty acid hydroperoxides without enzyme inactivation was developed.

Linoleic acid peroxidation by Solanum tuberosum lipoxygenase was activated in the presence of human 5-lipoxygenase-activating protein.

The present investigation describes the ability of human 5-lipoxygenase-activating protein (FLAP) to activate a plant 5-lipoxygenase. The presence of an active recombinant human FLAP in the 100000xg membrane fraction of infected Sf9 cells led to a specific increase in 9-hydroperoxyoctadecadienoic acid (9-HPOD) synthesis (+68%) or in 5-hydroperoxyeicosatetraenoic acid (5-HPETE) synthesis (+68%), after action of Solanum tuberosum tuber 5-lipoxygenase (S.t.LOX) on linoleic acid (natural plant lipoxygenase substrate) or on arachidonic acid. On the contrary, the presence of non-transfected membranes obtained from non-infected Sf9 cells led to an inhibition of lipoxygenase activity. MK-886, a potent inhibitor of leukotriene biosynthesis, blocked the FLAP dependent S.t.LOX activation after preincubation with FLAP transfected membranes. In conclusion, this study demonstrates that a recombinant human FLAP can stimulate a lipoxygenase other than mammalian 5-lipoxygenase (S.t.LOX) by using different polyunsaturated fatty acids as substrates.

Cortical cell elution by sedimentation field-flow fractionation.

As a cell sorter, Sedimentation field-flow fractionation (SdFFF) can be defined as an effective tool for cell separation and purification, respecting integrity and viability as well as providing enhanced recovery and purified sterile fraction collection. The complex cell suspension containing both neurons and glial cells of all types, obtained from cerebral cortices of 17-day-old rat fetuses, is routinely used as a model of primary neuronal culture. Using SdFFF, this complex cell mixture was eluted in sterile fractions which were collected and cultured. SdFFF cell elution was conducted under strictly defined conditions: rapid cell elution, high recovery (negligible cell trapping), short- and long-term cell viability, sterile collection. After immunological cellular type characterization (neurons and glial cells) of cultured cells, our results demonstrated the effectiveness of SdFFF to provide, in less than 6 min, viable and enriched neurons which can be cultured for further investigations.

Sedimentation field flow fractionation monitoring of rice starch amylolysis.

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this work, we investigated the capacity of SdFFF to monitor the native rice starch amylolysis. In order to determine if fractogram changes observed were correlated to granule biophysical modifications which occurred during amylolysis, SdFFF separation was associated with particle size distribution analysis. The results showed that SdFFF is an effective tool to monitor amylolysis of native rice starch. SdFFF analysis was a rapid (less than 10 min), simple and specific method to follow biophysical modifications of starch granules. These results suggested many different applications such as testing series of enzymes and starches. By using sub-population sorting, SdFFF could be also used to better understand starch hydrolysis mechanisms or starch granule structure.

Cyclooxygenase-2 up-regulation after FLAP transfection in human adenocarcinoma cell line HT29 cl.19A.

Five-lipoxygenase-activating protein (FLAP) is usually described as an essential protein to activate the leukotriene (LTs) synthesis via the 5-lipoxygenase pathway. In the enterocyte model HT29 cl.19A cell line, 5-lipoxygenase metabolism was found despite the lack of FLAP expression. Therefore HT29 cl.19A represents an original mammalian model to study FLAP-dependent leukotriene synthesis. In FLAP cDNA transfected HT29 cl.19A cells, FLAP expression led to an increase in cyclooxygenase pathway products (mainly PGE2) without an increase in 5-lipoxygenase metabolism. This increase in PGE2 synthesis was associated with a cyclooxygenase-2 upregulation in comparison to untransfected HT29 cl.19A cells. These results suggest a possible interaction between the two major pathways of arachidonic acid metabolism.

trans,trans-2,4-decadienal: cytotoxicity and effect on glutathione level in human erythroleukemia (HEL) cells.

The effects of trans,trans-2,4-decadienal (DDE), an isomer of a lipid peroxidation product were investigated on the human erythroleukemia cell line (HEL TIB 180). DDE strongly inhibits cell growth and affects cell viability without any differentiating effects. DDE treatment of HEL cells leads to a marked variation of the cellular glutathione level (GSH) and is involved in the beginning of DNA fragmentation.

Production of arachidonic acid metabolites by the colon adenocarcinoma cell line HT29 cl.19A and their effect on chloride secretion.

Eicosanoids were found in large amounts in the colonic mucosa of patients suffering from inflammatory bowel diseases and colonic adenocarcinoma. The aim of this study was to evaluate the role of the intestinal epithelial cells in the arachidonic acid metabolism and their functional response to certain eicosanoids. We used the human adenocarcinoma epithelial cell line HT29 cl.19A cell, which is an in vitro model of colon carcinoma and ion transport. These cells were found to express 5- and 15-lipoxygenase, leukotriene A4 hydrolase and cyclooxygenase-1 and -2 mRNAs. We observed an arachidonic acid metabolism via 5-lipoxygenase pathway despite the lack of FLAP mRNA expression and that certain eicosanoids such as hydroperoxy- and hydroxyeicosatetraenoic acids stimulate chloride secretion.

Chromatographic study of terpene derivatives on porous graphitic carbon stationary phase with beta-cyclodextrin as mobile phase modifier.

The stoichiometric coefficients and apparent formation constants (Kf) of alpha-terpineol, thymol, geraniol and linalool complexes with beta-cyclodextrin (beta-CD) were determined using HPLC with a porous graphitic carbon (PGC) chromatographic support. Measurements were performed with four different methanol-water mobile phases. All the terpene derivatives under study form 1:1 guest-CD complexes. Graphs of Kf as a function of the mobile phase composition appeared different from those classically described for RP-C18 and suggest that the PGC stationary phase could play an active role in the complexation process. Solute-CD inclusion and solute-stationary phase interactions may be involved in this specific behavior.

Sonication effect on cellular material in sedimentation and gravitational field flow fractionation.

Sonication procedures are generally used prior to field flow fractionation (FFF) separation in order to produce suspensions without aggregates. Yeast cells manufactured in active dry wine yeast (ADWY) were placed in an ultrasound water bath in order to disrupt possible clumps and to obtain a single-cell suspension to be used in optimal conditions during fermentation processes. In order to determine whether this sample preparation procedure meets absolute needs, different yeast samples before and after sonication were analysed by two field flow fractionation techniques. It is shown that 2 min of sonication in the sample preparation process is sufficient to obtain an optimal dispersion of the yeast cells, that is, without critical percentage of aggregates. To demonstrate this effect, photographs of the yeast cell suspensions were performed with non-sonicated and sonicated yeast sample dispersion. The resulting data are compared with the elution profiles obtained from the two different FFF techniques. It is demonstrated that fractogram profiles prove the effectiveness of sonication methodologies.

Deferred standards, an on-line qualification, validation and system stability probe for chromatographic assay.

Specific programming of automated HPLC systems allows total on-line qualification, validation and stability monitoring using the concept of deferred standards. Setting up such a process for routine analyses in an automated HPLC system requires specific autosampler programming as well as specific monitoring software. With an autosampler, a double injection procedure is programmed, the first introducing the sample, and the second, a few minutes deferred, the deferred control standard. Two additional compounds are therefore added to the sample before and during the chromatographic process: the intemal standard for sample quantification and the deferred standard for system control. Specific methodologies are described of how to obtain classical quantitative analysis information as well as system qualification validation stability information. Experiments were performed to develop specified methodologies to monitor the quality of quantitative analysis during the life of the column by using the deferred standard concept to probe the effects of column ageing on separation characteristics.

Resistance to apoptosis and cyclooxygenase-2 expression in a human adenocarcinoma cell line HT29 CL.19A.

BACKGROUND: COX-2 expression increases the tumorigenic potential of enterocytes. Tumorigenic effect is partially linked to an inhibition of programmed cell death which is one of the most important components in maintaining intestinal epithelium integrity. MATERIALS AND METHODS: We analyzed apoptosis in HT29 cl.19A cells cultured over 3 weeks, in the presence (10%) or in the absence of fetal bovine serum (FBS), by analysis of genomic DNA fragmentation after agarose gel electrophoresis, morphological measurement of apoptosis using DAPI chromatin staining, and transmission electron microscopy (TEM) to identify apoptotic cellular morphological changes. RESULTS: Regardless of the methods used, no apoptotic signs were observed for each culture condition, even if cells were cultured 3 weeks in the absence of FBS. CONCLUSION: Using HT29 cl.19A cells (untransfected cells), we found that intrinsic or constitutive COX-2 expression in adenocarcinoma cell line was associated with spontaneous resistance to apoptosis.