Partenaires

CNRS


Rechercher


Accueil > Publications > Recherche par années > Années 2000 > 2001

2001

Page(s) : < | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |

A heat labile soluble factor from Bacteroides thetaiotaomicron VPI-5482 specifically increases the galactosylation pattern of HT29-MTX cells

The aim of this work was to set up and validate an in vitro model to study a molecular response of an intestinal host cell line (HT29-MTX), to a non-pathogen microflora component We found that Bacteroides thetaiotaomicron strain VPI-5482 had the capacity to change a specific glycosylation process in HT29-MTX cells via a mechanism that involved a soluble factor. Differentiated HT29-MTX cells were grown in the presence of 20% of spent culture supernatant from the B. thetaiotaomicron during 10 days, Glycosylation processes were followed using a large panel of lectins and analysed using confocal microscopy, western blotting and flow cytometry techniques.

Lire la suite

A remarkably facile oxygen transfer in a nitrobenzofuroxan structure activated through sigma-complex formation

Treatment of 4,6-dinitrobetlzofuroxan (DNBF) with the imidazoline 1-NRf is found to afford a zwitterionic nitrogen-bonded complex (2-NRf) which, in the presence of base (Et3N), undergoes a slow but quantitative transformation to give 7-hydroxy-4,6-dinitrobmzofurazan (5) as the final product. Overall, an oxygen transfer has thus occurred from the N-oxide function to the carbocyclic moiety of DNBF. The key point in this transformation is shown to be a facile abstraction of the sp(3) hydrogen bonded at C-7 of 2-NRf, providing important new evidence that the parent DNBF structure is extremely electron-withdrawing (’super-electrophile’). The overall conversion is also an unusual case of a catalytic process in which the catalysts (both 1-NRf and Et3N) partake to form covalent reaction intermediates and thereby lower the activation energy, resulting in a facile reaction. (C) 2001 Elsevier Science Ltd. All rights reserved.

Lire la suite

Analysis of anti-HIV nucleoside inhibitors by capillary electrophoresis-electrospray ionization mass spectrometry

A method employing capillary electrophoresis (CE) with tandem mass spectrometry (MS) has been developed for the simultaneous determination, on one hand, of zidovudine (AZT) with stavudine (d4T), and on the other hand, of lamivudine (3TC) with a didanosine metabolite (ddA), four potent human immunodeficiency virus reverse transcriptase (RT-HIV) inhibitors. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) as well as the influence of magnesium cation upon electroosmotic flow, electrophoretic mobility and peak efficiency has been studied. The limit of detection (LOD) by this method is 2.5 ppb for AZT and 20 ppb for d4T, 2 ppb for ddA and 5 ppb for 3TC, respectively. This paper illustrates the current importance in CE-ESI/MS/MS technique as a complementary or substituted method to measure levels (at ng/mL) of anti-HIV drugs alone or in combination.

Lire la suite

Analysis of intracellular didanosine triphosphate at sub-PPB level using LC-MS/MS

An analytical procedure has been developed for the analysis of intracellular didanosine triphosphate (ddATP). An electrospray ionization tandem mass spectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a mobile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of the 5’-triphosphate metabolite of the antiviral didanosine. In this procedure. ddATP was extracted from CEM-T4 cells, isolated using an exchange anion solid phase extraction procedure, enzymatically dephosphorylated and then analyzed by LC-MS/MS within a I min run time.

Lire la suite

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes - Evidence of affinity for negatively charged membranes

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed Of L-a -1,2-dimyristoylphosphatidylcholine, L-a -1,2-dimyristoylphosphatidylglycerol and L-a -1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing L-a -1,2-dimyristoylphosphatidylglycerol ; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane.

Lire la suite

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen

Animals Vaccinated with heat shock protein (HSP)-peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP-peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96-peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques, Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates, Size distributions ranged from dimers to octamers and higher.

Lire la suite

Cardiac creatine kinase metabolite compartments revealed by NMR magnetization transfer spectroscopy and subcellular fractionation

In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstated by an apparent discrepancy between the forward (F-f) and reverse (F-r) CK flux if this compartment was neglected in the analysis [Joubert et al, (2000) Biophys. J. 79, 1-13], To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or ? ATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (F-f/F-r ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP), Under conditions masking the influence of the ATP-P-i exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy ; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential tool to assess metabolite compartmentation in the whole organ.

Lire la suite

Ceruloplasmin expression is highly increased in a transgenic mouse model of hepatocellular carcinoma

Transgenic mice expressing the Simian virus 40 large T antigen under the control of the liver-specific human antithrombin-III promoter all develop well-differentiated hepatocellular carcinoma. During tumour development serum ceruloplasmin (Cp) increases gradually until it reaches 30 times control levels in all transgenic mice at 6 months of age. The accumulation of Cp in the serum is due to the increased transcription of the Cp gene as well as to the increase in Cp mRNA stability in the livers of the transgenic mice. One-half of the overproduced Cp is charged with copper and Cp-associated serum oxidase activity increases in parallel with the holo-Cp concentration. Through its ferroxidase activity Cp is involved prominently in iron metabolism. Analysis of copper and iron in serum and liver revealed increased copper levels in the serum of tumour-bearing animals and which increased in parallel with Cp concentration ; the amounts of copper in the liver were unchanged. In contrast, serum iron remained constant during tumour development whereas the iron concentration in the livers of the transgenic mice decreased.

Lire la suite

Page(s) : < | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |