Synthesis, characterization and bio-functionalization of magnetic nanoparticles to improve the diagnosis of tuberculosis.

Mycobacterium tuberculosis is the cause of one of the diseases with the highest mortality and morbidity rate in the Americas and in the world. In developing countries, the diagnosis of tuberculosis (TB) is based on baciloscopy and bacteriological cultures. The first method has a low sensitivity, and the second can take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control this disease and favors the transmission of tuberculosis to the susceptible population. In this work, we present the synthesis, amine-silanization, characterization and bio-functionalization of magnetic nanoparticles (MNPs) to develop a sandwich ELISA to detect and concentrate antigens from M. tuberculosis. For this purpose, a recombinant mycobacterial heat shock protein Hsp16.3, which contributes to the persistence of TB, was cloned and expressed in the E. coli system. Polyclonal antibodies anti-Hsp16.3 were produced in a rabbit and in mice. Magnetic nanoparticles were synthesized by co-precipitation, amine-functionalized and characterized by several physical-chemical methods. The XRD, Mossbauer spectroscopy, zeta potential, TEM, and FTIR all proved the successful preparation of the MNPs showing a diffraction crystal diameter of 10.48 ± 2.56 nm, superficial net charge of [Formula: see text]: +23.57 ± 2.87 mV, characteristic patterns of magnetite and a structure similar to a sphere. Additionally, it showed a magnetization saturation of 37.06 emu.g-1. For the functionalization of nanoparticle surfaces with anti-Hsp16.3, the active ester method was used for bond formation, and parameters such as time of incubation, coupling agents ratio (EDC/NHS) and concentration as well as surface saturation level of amine-silanized MNPs (MNP@Si@NH2) were standardized. Finally, bio-functionalized MNPs were used to detect, fix and concentrate the recombinant antigen Hsp16.3 from M. tuberculosis in a sandwich ELISA-MNP assay.

Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain highlights a local metabolic regulation after oral versus subcutaneous administration.

17ß-Estradiol (17ß-E2) is a steroid with pleiotropic actions. In addition to being a sexual hormone, it is also produced in the brain where it modulates the reproductive axis. It has been shown that 17ß-E2 also acts on synaptic plasticity and plays a role in neurological pathways and in neurodegenerative diseases. Assaying this steroid in the brain is thus interesting to improve our knowledge of 17ß-E2 effects in the brain. However, 17ß-E2 concentration in the central nervous system has been reported to be of a few nanograms per gram wet weight (nanomolar range concentration); therefore, its quantification requires both an efficient extraction process and a sensitive detection method. Herein is presented a derivatization-free procedure based on solid-phase extraction followed by LC-MS/MS analysis, targeted on 17ß-E2, its isomer17α-E2, and its metabolites estrone (E1) and estriol (E3). This extraction process allowed reaching 96% 17ß-E2 recovery from the mouse brain. Limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 2.5 pmol mL-1, respectively, were reached for both 17α-E2 and 17ß-E2. LOD values for E1 and E3 were 0.01 and 0.025 pmol mL-1, respectively. The variation coefficients for intra- and inter-assays were 6 and 14%, respectively, for both estradiol forms. The method was applied to assess estrogen levels in the mouse brain and hippocampus after 17ß-E2 acute (subcutaneous injection) and chronic (drinking water) physiological administration. Total estrogen levels were determined after enzymatic deconjugation and compared to free estrogen levels. While 17α-E2 was not detected in biological samples, 17ß-E2 and metabolite measurements highlight a local biotransformation of estrogens after physiological administration via drinking water. Graphical abstract Method workflow: After oral or subcutaneous Estradiol administration, mouse brain or hippocampus was removed. Samples were homogenized and prepared according to a liquid-liquid extraction, followed by a solid-phase extraction. Then, LC-MS/MS was optimized to quantify 17ß-E2, its isomer17α-E2, its metabolites estrone (E1) and estriol (E3) and their conjugates.

Flavanone metabolites decrease monocyte adhesion to TNF-α-activated endothelial cells by modulating expression of atherosclerosis-related genes.

Flavanones are found specifically and abundantly in citrus fruits. Their beneficial effect on vascular function is well documented. However, little is known about their cellular and molecular mechanisms of action in vascular cells. The goal of the present study was to identify the impact of flavanone metabolites on endothelial cells and decipher the underlying molecular mechanisms of action. We investigated the impact of naringenin and hesperetin metabolites at 0·5, 2 and 10 µM on monocyte adhesion to TNF-α-activated human umbilical vein endothelial cells (HUVEC) and on gene expression. Except hesperetin-7-glucuronide and naringenin-7-glucuronide (N7G), when present at 2 µM, flavanone metabolites (hesperetin-3'-sulphate, hesperetin-3'-glucuronide and naringenin-4'-glucuronide (N4'G)) significantly attenuated monocyte adhesion to TNF-α-activated HUVEC. Exposure of both monocytes and HUVEC to N4'G and N7G at 2 µM resulted in a higher inhibitory effect on monocyte adhesion. Gene expression analysis, using TaqMan Low-Density Array, revealed that flavanone metabolites modulated the expression of genes involved in atherogenesis, such as those involved in inflammation, cell adhesion and cytoskeletal organisation. In conclusion, physiologically relevant concentrations of flavanone metabolites reduce monocyte adhesion to TNF-α-stimulated endothelial cells by affecting the expression of related genes. This provides a potential explanation for the vasculoprotective effects of flavanones.

Identification, Quantitation, and Sensory Evaluation of Thiols in Bordeaux Red Wine with Characteristic Aging Bouquet.

Great Bordeaux red wines are known for their distinctive aging bouquet. However, the nature of volatile chemicals underpinning this sensory expression is not fully understood. This work investigated the empyreumatic aging bouquet of a collection of premium Bordeaux red wines using silver-ion (Ag+) solid-phase extraction, cryogenic heart-cutting multidimensional gas chromatography mass spectrometry/olfactometry, and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. In doing so, a substantial number of "meaty" odors were revealed. Three detected "meaty" notes were tentatively or unequivocally attributed to furan thiols. Among them, 2-methyltetrahydrofuran-3-thiol (1) with a pleasant "meaty" aroma was reported in wine for the first time. Its trans isomer (trans-1a) was resolved from its racemate by chemical modification, which confirmed its presence in wine. The odor detection threshold of trans-1a in the model wine was determined at 55 ng/L. Moreover, an additive effect between 1 and literature-known 2-methyl-3-furanthiol was observed. By a new ultra high-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry method, the concentration of trans-1a, in addition to those of 2-methyl-3-furanthiol and 2-furfuryl thiol, was measured in the wines at ng/L levels.

Identification and Distribution of New Impact Aldehydes in Toasted Oak Wood (Quercus petraea).

The purpose of this study was to identify aroma compounds associated with the toasting intensity of oak wood (Quercus petraea). Crude organic extracts from oak wood samples (toasted at different temperature-time couples) were analyzed by a sensory-guided approach using GC-O-TOFMS, followed by purification with semipreparative HPLC (reverse phase). This approach revealed two specific odorous zones (OZs) reminiscent of metal and puff pastry. The first OZ was identified as trans-4,5-epoxy-(E)-2-decenal (1) by coinjection of the commercial product, whereas identification of (2E,4E,6Z)-nonatrienal (2) associated with puff pastry OZ was validated by a multistep chemical synthesis approach (Wittig reaction) followed by semipreparative HPLC purification (chiral phase). Their detection thresholds in model wine solution were 60 ng/L (1) and 16 ng/L (2). Their distribution in toasted oak wood samples [GC-NCI-MS (NH3) analysis] ranged from some ng/g to 210 ng/g for (1) and 85 ng/g for (2). Finally, additional sensory experiments demonstrated the impact of newly identified aldehydes in toasted oak wood.

Biorecognition and detection of antigens from Mycobacterium tuberculosis using a sandwich ELISA associated with magnetic nanoparticles.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the 10 leading causes of death worldwide, especially in low-income areas. A rapid, low-cost diagnostic assay for TB with high sensitivity and specificity is not currently available. Bio-functionalized magnetic nanoparticles (MNPs) which are able to efficiently detect and concentrate biomolecules from complex biological samples, allows improving the diagnostic immunoassays. In this way, a proof-of-concept of MNP-based sandwich immunoassay was developed to detect various MTB protein antigens. The superficial and secretory antigenic proteins considered in this research were: CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B, and MoeX. The proteins were cloned and expressed in an E. coli system. Polyclonal antibodies (ab) against the recombinant antigens were elicited in rabbits and mice. Antibodies were immobilized on the surface of amine-silanized nanoparticles (MNP@Si). The functionalized MNP@Si@ab were tested in a colorimetric sandwich enzyme-linked immunosorbent assay (sELISA-MNP@Si@ab) to recognize the selected antigens in sputum samples. The selected MTB antigens were successfully detected in sputum from TB patients in a shorter time (~ 4 h) using the sELISA-MNP@Si@ab, compared to the conventional sELISA (~15 h) standardized in home. Moreover, the sELISA-MNP@Si@ab showed the higher sensitivity in the real biological samples from infected patients.

Aromatic Potential of Bordeaux Grape Cultivars: Identification and Assays on 4-Oxononanoic Acid, a γ-Nonalactone Precursor.

γ-Nonalactone has been demonstrated to be a chemical marker of dried/cooked fruit nuances detected in must and wine, but little is known about its formation pathways. Therefore, on the basis of the literature, we hypothesized 4-oxononanoic acid as a potential precursor. Using dichloromethane extraction followed by gas chromatography coupled to negative chemical ionization mass spectrometry, this keto acid was identified and quantified in Merlot and Cabernet Sauvignon musts. Its concentration ranged from traces to 60 µg/L. The biotransformation of 4-oxononanoic acid into γ-nonalactone by Saccharomyces cerevisiae during alcoholic fermentation was demonstrated using labeled d6-4-oxononanoic acid. Additional experiments shed light on the 4-oxononanoic acid role as a γ-nonalactone precursor and revealed that this biotransformation was (R)-enantioselective. Sensory and distribution studies of the enantiomers revealed that the detection threshold of R and S forms were 66 and 35 µg/L and the average ratio of R/S in grape and wine was 94:6 and 65:35.

Quantitation, Organoleptic Contribution, and Potential Origin of Diethyl Acetals Formed from Various Aldehydes in Cognac.

Cognac wine distillate (WD), especially that produced during aging, is marked by complex and elegant aroma. This work aimed at expanding the knowledge on the Cognac WD aroma by a sensory-guided approach, involving a fractional-distillation technique and gas chromatography coupled to olfactometry and mass spectrometry (GC-O-MS). In doing so, a fruity-odor zone was highlighted in WD extracts that was attributed to the diethyl acetal family. Ten additional diethyl acetals were detected by GC-MS. Next, an assay method was developed and validated for seven of these diethyl acetals. Their detection thresholds were evaluated in a model solution of water/ethanol (60:40, v/v). 1,1-Diethoxy-3-methylbutane was shown to present a significant organoleptic impact because its olfactory-detection threshold (323 µg/L) is lower than its range of concentrations in WD (461 to 3337 µg/L). Given that diethyl acetals result from reactions between ethanol and aldehydes, quantitative correlations between diethyl acetals and corresponding aldehydes were considered.

Identification of Dialkylpyrazines Off-Flavors in Oak Wood.

Volatile extractive compounds from high-quality oak wood (Quercus sp.) are responsible for important pleasant olfactory notes, such as coconut, wood, vanilla, caramel, and spice. Recently, a new off-flavor reminiscent of rancid butter has been detected in oak wood. Using gas chromatography-olfactometry (GC-O) coupled to several detection modes, such as nitrogen-phosphorus detection (GC-O-NPD) or mass spectrometry (GC-O-MS) and multidimensional GC-O coupled to time-of-flight mass spectrometry, six compounds containing nitrogen atoms were identified. The volatiles were suggested to belong to 2,5-disubstituted pyrazines family, which was confirmed by comparison with synthetic reference compounds. For this purpose, symmetric and dissymmetric 2,5-dialkylpyrazines were prepared from methyl esters of corresponding aliphatic amino acids (Val, Leu, and Ile) by a three-step, one-pot reaction under mild reducing conditions. Organoleptic descriptors and odor detection thresholds were also determined, whereas a bacterial origin explaining these off-flavors was hypothesized.

Analysis of the diastereoisomers of the cysteinylated aroma precursor of 3-sulfanylhexanol in Vitis vinifera grape must by gas chromatography coupled with ion trap tandem mass spectrometry.

The diastereoisomeric distribution of S-3-(hexan-1-ol)cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexan-1-ol (3SH) in Vitis vinifera grape juice, was determined by a new method. This procedure is based on the purification of P-3SH in a small volume of must (500 microL) by affinity chromatography, followed by the separation of chiral molecules in derivative forms by gas chromatography coupled with ion trap tandem mass spectrometry (GC-MS/MS). The diastereoisomers were easily separated using heptafluorobutyric anhydride and heptafluorobutanol (HFBA and HFOH) as derivatization reagents. Method validation was conducted using samples of grape juice, synthetic must, fermenting must, and wine that were fortified with P-3SH at concentrations of 0.6 and 2.5 microM. The relative standard deviation (RSD) and limit of detection (LOD) of the GC-MS/MS method were 4.6% and 1.5 nM, respectively. P-3SH assays in Bordeaux white grape juice affected by Botrytis cinerea showed an unusually increased proportion of the RS form of the precursor (approximately RR:RS=30:70) as compared to a diastereoisomer ratio (in the vicinity of 50:50) in healthy grape juice.

Original preparation of conjugates for antibody production against Amicoumacin-related anti-microbial agents.

Amicoumacins are natural products with potent anti-ulcerogenic and anti-bacterial activities, and have been isolated from different Bacillus genera. They belong to a family of 3,4-dihydroisocoumarin derivatives bearing hydroxylated amino acid side chains. The 3,4-dihydroisocoumarin moiety of Amicoumacins has been obtained in two steps from 2-methoxybenzoic acid by combining directed and benzylic metalation strategies. The use of s-BuLi in both steps gave satisfactory and reproducible yields. For the development of an immunoassay (ELISA) of Amicoumacin-related compounds in biological media, the deprotected 3,4-dihydroisocoumarin moiety has been coupled to the BSA carrier protein via a homobifunctional linker deriving from d-tartaric acid. This approach enabled to introduce the hydroxylated portion of Amicoumacin directly during the preparation of hapten-protein conjugates. The coupling ratio was evaluated by mass spectrometry. The hapten-protein conjugate showing the best coupling ratio was used to generate polyclonal immunosera in rabbits. After immunoserum titration, ELISA conditions were set up and specificity tests were performed on solutions of pure parent compounds, semi-purified Amicoumacin B as well as on culture supernatants of strains known for their Amicoumacin production. This immunoassay is suitable for a rapid and simple screening test for the production of Amicoumacins and its related compounds by bacterial strains.

Surprising structural lability of a cysteine-S-conjugate precursor of 4-methyl-4-sulfanylpentan-2-one, a varietal aroma in wine of Vitis vinifera L. cv. Sauvignon blanc.

4-Methyl-4-sulfanylpentan-2-one (1; 4MSP) provides a characteristic aroma compound of wines made from Vitis vinifera L. cv. Sauvignon blanc. 4MSP has a strong box-tree odor with a very low perception threshold and is derived from the cysteinylated precursor S-(1,1-dimethyl-3-oxobutyl)cysteine (4; P-4MSP). P-4MSP is transformed into 4MSP during alcoholic fermentation and is an excellent marker of varietal aroma potential. An improved synthesis of P-4MSP as well as of its deuterium-labeled analogue [D(6)]-P-4MSP is described. Several analytical methods (NMR, IR, LSI-MS, GC/MS, ESI-MS(n)) were combined to elucidate spontaneous reversible structural changes of P-4MSP at different pH values. At low pH, P-4MSP has a linear keto form. The keto-enol tautomerism was observed at neutral pH. At pH 8, the formation of N-substituted intramolecular hemiaminal was characterized by ESI-MS and ESI-MS(n) experiments. The hemiaminal loses H(2)O at high pH to produce a cycloimine, which is easily opened by acid hydrolysis. The keto-enol tautomerism explained the incorporation of only six D-atoms during the preparation of the P-4MSP deuterated standard even if [D(10)]mesityl oxide was used. Derivatization conditions for GC/MS analysis strongly affected the ratio of the monosilylated intramolecular cyclic form and the disilylated linear form of P-4MSP. The structural changes of P-4MSP may have a considerable impact on the development of methods of measuring varietal aroma potential.

Odorous impact of volatile thiols on the aroma of young botrytized sweet wines: identification and quantification of new sulfanyl alcohols.

Specific extraction of volatile thiols using sodium p-hydroxymercuribenzoate revealed the presence of three new sulfanylalcohols in wines made from Botrytis-infected grapes: 3-sulfanylpentan-1-ol (II), 3-sulfanylheptan-1-ol (III), and 2-methyl-3-sulfanylbutan-1-ol (IV). The first two have citrus aromas, whereas the third is reminiscent of raw onion. In addition, 2-methyl-3-sulfanylpentan-1-ol, which has a raw onion odor, was tentatively identified. Like 3-sulfanylhexan-1-ol (I), already reported in Sauternes wines, compounds II, III, and IV were absent from must. They were found in wine after alcoholic fermentation, and their concentrations were drastically higher when Botrytis cinerea had developed on the grapes. In the commercial botrytized wines analyzed, the mean levels of II, III, and IV were 209, 51, and 103 ng/L, respectively. Despite their low odor activity values, sensory tests showed additive effects among I, II, and III, thus confirming their olfactory impact on the overall aroma of botrytized wines.

Self-tuning interfacial architecture for Estradiol detection by surface plasmon resonance biosensor.

This study reports the operation principles for reusable SPR biosensors utilizing nanoscale-specific electrostatic levitation phenomena in their sensitive layer design. Functional macromolecular building blocks localized near the "charged" surface by a variety of weak electrostatic interactions create a flexible and structurally variable architecture. A proof-of-concept is demonstrated by an immunospecific detection of 17ß-Estradiol (E2) following the competitive inhibition format. The sensing interfacial architecture is based on the BSA-E2 conjugate within the BSA matrix immobilized on the "charged" (as a result of guanidine thiocyanate treatment) gold surface at pH 5.0. Kinetic analysis for different E2 concentrations shows that using parameter ß of the stretched exponential function ~(1-exp(-(t/τ)ß) as an analyte-specific response measure allows one to substantially decrease the low detection limit (down to 10-3ng/ml) and increase the dynamic range (10-3-103ng/ml) of the SPR biosensor. Finally, it's concluded that the created interfacial architecture is a typical complex system, where SPR response is formed by the stochastic interactions within the whole variety of processes in the system. The E2 addition destroys the uniformity of the reaction space (where an interaction of the antibody (Ab) and the analog of E2 in the self-tuneable matrix takes place) by the redistribution of the immunospecific complexes Ab(E2)x (x=0, 1, 2) dependent on E2 concentration. Binding dynamics changes are reflected in the values of ß which summarize in compact form all "hidden" information specific for the evolving distributed interfacial system.

Identification and Organoleptic Contribution of Vanillylthiol in Wines.

Vanillylthiol, a chemical compound reminiscent of clove and smoke, has been identified for the first time in young red and dry white wines. The chemical structure of this new aroma was confirmed by original chemical synthesis. Vanillylthiol was prepared by a two-step procedure from vanillin. The conversion of vanillin to divanillyl disulfide was easily achieved by treatment with an inorganic sulfur-donor reagent. Reduction of the disulfide gave the target thiol in good yield. The quantification of vanillylthiol in wine was performed by nonspecific liquid/liquid extraction (CH2Cl2), separation of the volatile compounds using gas chromatography, and specific detection using tandem mass spectrometry (triple quadrupole). Vanillylthiol was found particularly in young wines aged in new oak barrels. These wines contained between a few 50 ng/L to more than 8300 ng/L. The highest levels were found in red wines aged 12 months in new oak barrels. Given its perception threshold in a wine model solution (3.8 µg/L), vanillylthiol may contribute to the spicy, clove-like flavor of red wines aged in oak barrels.

Identification of S-3-(hexanal)-glutathione and its bisulfite adduct in grape juice from Vitis vinifera L. cv. Sauvignon blanc as new potential precursors of 3SH.

Two main precursors (S-3-(hexan-1-ol)-l-cysteine and S-3-(hexan-1-ol)-l-glutathione) of 3-sulfanylhexanol (3SH, formerly named 3-mercaptohexanol) have been identified so far in grape juice but a correlation between precursor concentrations in grape juices and 3SH concentrations in wines is not always observed. This suggests that there may be other compounds associated with the aromatic potential. In this work, S-3-(hexanal)-glutathione (Glut-3SH-Al) and its bisulfite (Glut-3SH-SO3) adduct were identified in Sauvignon blanc grape juice by liquid chromatography coupled to Fourier transform mass spectrometry experiments. A partial purification of the compounds was carried out by Medium Pressure Liquid Chromatography (MPLC) on the reverse phase using 5L of grape juice. The addition of synthetized Glut-3SH-Al and Glut-3SH-SO3 in the synthetic medium induced a significant release of 3SH after fermentation. Moreover, we demonstrate that Glut-3SH-Al and its bisulfite adduct are present in grape juice and could be considered as new direct 3SH precursors with molar conversion yields close to 0.4%.

Design and validation of a novel immunological test for enterolactone.

Enterolactone (ENL) is produced by the gut microflora from lignans found in edible plants. ENL is estrogenic with no effect on the E-screen test and is a natural Selected Estrogen Receptor Modulator (SERM) with health interests that have to be checked in clinical studies with bioavailability assessment. Two haptens of ENL were synthesized, with a spacer arm at the C5 position having either 2 or 4 carbon atoms (ENLΔ2 and ENLΔ4, respectively). Hapten coupling to bovine serum albumin (BSA) was characterized by MALDI mass spectrometry. Polyclonal antibodies were obtained against the BSA conjugates. Additional conjugates were generated by coupling to swine thyroglobulin (Thyr). Homologous and heterologous competitive ELISAs were developed with Thyr or BSA conjugates as coating. The best assays were validated on biological samples from mice. Both antibodies exhibited the same IC50 at 1.5 ng mL(-1) with a detection limit below 0.5 ng mL(-1). Most cross-reactions with structurally related lignans were lower than 0.03%. This new assay type is faster, more specific and more reliable than existing ones.

Bioavailability of glycitein relatively to other soy isoflavones in healthy young Caucasian men.

Glycitein is a Selective Estradiol Receptor Modulator (SERM) from soy. The study reports plasma bioavailability and urine excretion of glycitein compared to other soy isoflavones after a unique intake of food supplement based on soy germ containing 55.24mg isoflavones. Eighteen plasma and urinary sampling profiles collected over 48h from healthy young Caucasian men were analysed using specific ELISAs. Eight profiles contained equol. Glycitein T(max), C(max), AUC(0→24h) and T(½) in plasma were calculated. Urine T(max), % of excretion at 24h and clearance were assessed. Glycitein is one of the best absorbed flavonoids. Plasma steady-state level can be achieved by several intakes a day. Glycitein bioavailability is similar to that of daidzein and its urinary excretion is significantly higher than that of genistein. Equol does not affect glycitein bioavailability. Knowing glycitein bioavailability in man is essential for the development of soy-germ-based food supplements for health applications.

The effect of low pH on the glycitein-BSA conjugate interaction with specific antiserum: competitive inhibition study using surface plasmon resonance technique.

Competitive inhibition serological assay for detection of the phytoestrogen glycitein (Glyc) was developed using surface plasmon resonance (SPR) technique with protein conjugates and polyclonal antibodies initially designed for the enzyme-linked immunosorbent assays (ELISA). The efficiency of the approach to the quantification of the soy isoflavone glycitein in water was investigated using the competitive reaction of analyte (free Glyc)and immobilized Glyc-BSA-conjugate with polyclonal antibodies. It was shown that the efficiency to detect Glyc drastically depends on the pH level of the probe solution. With the decrease in pH from 7.4 to 4.0, (i) the affinity of the specific reaction increases and (ii) the level of unspecific sorption becomes saturated. Non-specific adsorption to a SPR sensor surface obscures the specific component and shaded specific response at higher pH (6.0-7.4) when used serum for the quantification of specific analytes. The standard curves obtained in acidic solutions (pH 4-5) indicate that the linear part of the dependence completely covers the range between detection limit (0.1 µg/ml) and Glyc solubility in water (0.9 µg/ml). The difference in SPR- and ELISA-based analytical protocols as well as the requirements for increasing the efficiency in quantitative SPR analysis using purified antibodies is discussed.

Membrane mineralocorticoid but not glucocorticoid receptors of the dorsal hippocampus mediate the rapid effects of corticosterone on memory retrieval.

This study was aimed at determining the type of the glucocorticoid membrane receptors (mineralocorticoid receptors (MRs) or glucocorticoid receptors (GRs)) in the dorsal hippocampus (dHPC) involved in the rapid effects of corticosterone or stress on memory retrieval. For that purpose, we synthesized corticosterone-3-O-carboxymethyloxime-bovine serum albumin conjugate (Cort-3CMO-BSA) conjugate (a high MW complex that cannot cross the cell membrane) totally devoid of free corticosterone, stable in physiological conditions. In a first experiment, we evidenced that an acute stress (electric footshocks) induced both a dHPC corticosterone rise measured by microdialysis and memory retrieval impairment on delayed alternation task. Both the endocrinal and cognitive effects of stress were blocked by metyrapone (a corticosterone synthesis inhibitor). In a second experiment, we showed that bilateral injections of either corticosterone or Cort-3CMO-BSA in dHPC 15 min before memory testing produced impairments similar to those resulting from acute stress. Furthermore, we showed that anisomycin (a protein synthesis inhibitor) failed to block the deleterious effect of Cort-3CMO-BSA on memory. In a third experiment, we evidenced that intra-hippocampal injection of RU-28318 (MR antagonist) but not of RU-38486 (GR antagonist) totally blocked the Cort-3CMO-BSA-induced memory retrieval deficit. In a fourth experiment, we demonstrated that RU-28318 administered 15 min before stress blocked the stress-induced memory impairments when behavioral testing occurred 15 min but not 60 min after stress. Overall, this study provides strong in vivo evidence that the dHPC membrane GRs, mediating the rapid and non-genomic effects of acute stress on memory retrieval, are of MR but not GR type.