Instrumental and sensory approaches for the characterization of compounds responsible for wine aroma

More than 800 aromatic compounds have been identified in wine, some of them at the ng/l level. Wine, therefore, constitutes a very complex matrix, from which it is difficult to isolate a specific aroma character. Gas chromatography-olfactometry (GC-O) applied to wine extracts is used to characterize odor-active zones that are often treated in a hierarchical way by Aroma Extract Dilution Analysis (AEDA). The aromatic impact of the volatiles is evaluated, generally by determining perception thresholds. This methodology has provided convincing results concerning wine flavors, but it does have its limitations. For instance, data on beta-damascenone have demonstrated that these methods could reach their limits for this volatile, in particular, because of the non-quantitative representation of aroma extracts of wines, and because of the difficulty to accurately determine the perception threshold in wines for a compound already present. For beta-damascenone, we have shown that its very low detection threshold with GC-O, its wide range, and its dependence on the composition of the medium resulted in overestimating its direct impact on the aroma of wine. Another way to facilitate the characterization of aromatic compounds was, therefore, investigated. High-Performance Liquid Chromatography (HPLC) methods were developed for the analysis of wine extracts. From an aromatic extract, 25 fractions with various flavors were thus obtained, and reverse-phase methodology was used for the selection and characterization of red- and black-fruit aromas in red wines.     

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARalpha-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARalpha independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.     

Lactobacillus casei DN-114 001 inhibits the ability of adherent-invasive Escherichia coli isolated from Crohn's disease patients to adhere to and to invade intestinal epithelial cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

The MRP1-mediated effluxes of arsenic and antimony do not require arsenic-glutathione and antimony-glutathione complex formation

Arsenic trioxide is an effective treatment for acute promyelocytic leukemia, but resistance to metalloïd salts is found in humans. Using atomic absorption spectroscopy, we have measured the rate of uptake of arsenic trioxide and of antimony tartrate in GLC4 and GLC4/ADR cells overexpressing MRP1 and the rate of their MRP1-mediated effluxes as a function of the intracellular GSH concentration. In sensitive cells, after 1 h, a pseudosteady state is reached where intra- and extracellular concentrations of metalloid are the same. This precludes the formation, at short term, of complexes between arsenic or antimony with GSH. In resistant cells reduced intracellular accumulation of arsenic (or antimony), reflecting an increased rate of arsenic (or antimony) efflux from the cells, is observed. No efflux of the metalloid is observed in GSH depleted cells. The two metalloïds and GSH are pumped out by MRP1 with the same efficiency. Moreover for the three compounds 50% of the efflux is inhibited by 2 M MK571. This led us to suggest that As- and Sb-containing species could be cotransported with GSH.     

Synthesis,characterization, cytotoxic activity,and cellular accumulation of dinuclear platinum complexes derived from N,N'-di-(2-aminoethyl)-1,3-diamino-2-propanol,aryl substituted N-benzyl-1,4-butanediamines,and N-benzyl-1,6-hexanediamines

This work describes the synthesis and characterization of six new dinuclear platinum complexes having N,N'-di-(2-aminoethyl)-1,3-diamino-2-propanol, aryl substituted N-benzyl-1,4-butanediamines and N-benzyl-1,6-hexanediamines as ligands. They were prepared by the reaction of cis-[PtCl(2)(DMSO)(2)] (DMSO=dimethyl sulfoxide) with the appropriate ligand in water, except for one of them, which was prepared from K(2)PtCl(4). We also report the cytotoxic activity and cellular accumulation of three of these complexes in a human small-cell lung carcinoma cell line and its resistant subline. Resistant cells exhibited a lesser degree of cross-resistance to these compounds when compared to cisplatin. The accumulation of platinum in both cell lines followed the same pattern, i.e. approximately the same intracellular platinum concentration yielded the same cytotoxic effect independent of the nature of the platinum complex used.     

Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1)

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.     

New insights into the rat spermatogonial proteome: identification of 156 additional proteins

Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.     

Ecological,Dynamic and Taxonomic Problems Due to <Emphasis Type="Italic">Ludwigia</Emphasis> (Onagraceae) in France

We analyzed meta-community structure of bdelloid rotifers colonizing mosses along an 80 meter section of Rio Valnava in NW Italy. Bdelloid rotifers are small animals living associated with a substratum; colonization in bdelloids can be produced by active animals moving along the riverbed, or by passive dormant propagules, moved by wind. To detect which kind of colonization might be stronger at different spatial scales, we designed a spatially nested sampling experiment at three hierarchical levels: (1) single sample, (2) 10 communities inside each pool, (3) complete section of 10 pools. Assessing species richness and species similarity of communities, and coherence and nestedness of bdelloid meta-communities, we found that different forces may drive species composition at different spatial scales: at the largest scale, colonization of propagules may over-ride direct dispersal between pools, while at the scale of the single pool, differential movements of species give a nested structure to the meta-communities. The number of species increased as the level of analysis increased, even though this study was carried out along only a small stream section.     

Osmotic stress, a proinflammatory signal in Caco-2 cells

Hyper- (450 mOsm/l) and hypoosmotic exposure (150 mOsm/l) of Caco-2 cells, a human intestinal epithelial cell line, induced a twofold- and a fivefold increase in the production of IL-8, a constitutively expressed cytokine, respectively. This was observed both in the presence or in the absence of added proinflammatory cytokines and the stimulatory effect of osmotic stress was additive to that induced by the cytokines. Thus, IL-8 production appeared minimal around isoosmolarity, i.e. 300 mOsm/l. Concerning the signalling pathway involved, specific inhibition of p38- or p42/44 MAP kinases decreased the IL-8 production by about 30% independently of the osmotic condition used. Inhibition of c-jun-NH2-terminal kinase (JNK) by using both dicoumarol and SP600125 totally inhibited the stimulatory effect of hypoosmolarity. Moreover, hypoosmolarity induced an about threefold increase in JNK activity demonstrating that JNK was specifically involved in the effect of hypoosmolarity on IL-8 production. This is not the case for hyperosmolarity. Such an effect of osmotic stress was not restricted to IL-8, but was also observed on the production of IL-6, a non-constitutively expressed cytokine. Again, IL-6 production appeared minimal in isoosmotic condition. Taken together, these results demonstrate that osmotic stress is a proinflammatory signal in Caco-2 cells and suggest that an osmosensor might specifically exist in intestinal epithelial cells.     

Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis

The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1–16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k ₊=0.3–1.4 s⁻¹ and k _off=0.05–0.25 s⁻¹, respectively). The bond distribution is binomial, with an average bond number ≤10 at these time scales. Increasing the fibronectin density (100–3000 molecules/μm²) raised k ₊ in a diffusion-dependent manner, leaving k _off relatively unchanged. Increasing the temperature (23–37 °C) raised both k ₊ and k _off, allowing calculation of the activation energy of the chemical reaction (around 20 k _B T). Increasing the compressive force on the cell during contact (up to 60 pN) raised k ₊ in a logarithmic manner, probably through an increase in the contact area, whereas k _off was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond. Received: 2 November 1999 / Revised version: 6 March 2000 / Accepted: 19 April 2000