Oxidative insults are associated with apolipoprotein E genotype in Alzheimer's disease brain.

The epsilon4 allele of the apolipoprotein E gene (APOE) is associated with sporadic and familial late-onset Alzheimer's disease (AD). Oxidative stress is believed to play an important role in neuronal dysfunction and cell death in AD. We now provide evidence that in the hippocampus of AD, the level of thiobarbituric acid-reactive substances (TBARS) and the APOE genotype are linked. Within AD cases, the levels of TBARS were found to be higher among epsilon4 carriers while the apoE protein concentrations were lower. The relationship between the levels of TBARS and apoE proteins was corroborated by the results from the APOE-deficient mice, in which the levels of TBARS were higher than those in wild-type mice. Among AD cases, tissues from patients with the epsilon4 allele of APOE displayed lower activities of catalase and glutathione peroxidase and lower concentration of glutathione than tissues from patients homozygous for the epsilon3 allele of APOE. Together these data demonstrate that, in AD, the epsilon4 allele of APOE is associated with higher oxidative insults.

Structural requirements of 5-hydroxytryptamine-moduline analogues to interact with the 5-hydroxytryptamine1B receptor.

5-Hydroxytryptamine-moduline is an endogenous cerebral tetrapeptide that regulates the activity of 5-hydroxytryptamine1B receptors. Direct binding of 5-[3H]hydroxytryptamine-moduline on rat brain homogenate evidenced the existence of two interacting sites for the peptide, very likely corresponding to different conformations of the 5-hydroxytryptamine1B receptor: The peptide first binds to a low-affinity state of the receptor (pIC50 = 7.68+/-0.14) and then induces (or stabilizes) a high-affinity complex (pIC50 = 11.62+/-0.18). This work focuses on the ability of 5-hydroxytryptamine-moduline analogues to recognize the high- and low-affinity sites for 5-hydroxytryptamine-moduline. The results obtained show that the two conformers of the 5-hydroxytryptamine1B receptor have similar but not identical binding pockets for 5-hydroxytryptamine-moduline. These two sites proved to be stereoselective and selective for tetrapeptides and favored the binding of peptides with hydrophobic amino acids in positions 1 and 4, serine in position 2, and a short amino acid in position 3. However, the serine in position 2 seems to be more important for the interaction of the peptide with the low-affinity site than the high-affinity one, which only needs a short hydrophobic amino acid in position 2.

Endoproteolytic activity in mammalian brain membranes cleaves 5-hydroxytryptamine-moduline into dipeptides.

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [3H]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn2+ ions and a pH optimum in the 7-8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a Km of 37.1 +/- 3.6 microM and a Vmax of 5.5 micromol min(-1) l(-1) per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726-735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.

Optimization of cardiac fiber orientation for homogeneous fiber strain during ejection.

The strain of muscle fibers in the heart is likely to be distributed uniformly over the cardiac walls during the ejection period of the cardiac cycle. Mathematical models of left ventricular (LV) wall mechanics have shown that the distribution of fiber strain during ejection is sensitive to the orientation of muscle fibers in the wall. In the present study, we tested the hypothesis that fiber orientation in the LV wall is such that fiber strain during ejection is as homogeneous as possible. A finite-element model of LV wall mechanics was set up to compute the distribution of fiber strain at the beginning (BE) and end (EE) of the ejection period of the cardiac cycle, with respect to a middiastolic reference state. The distribution of fiber orientation over the LV wall, quantified by three parameters, was systematically varied to minimize regional differences in fiber shortening during ejection and in the average of fiber strain at BE and EE. A well-defined optimum in the distribution of fiber orientation was found which was not significantly different from anatomical measurements. After optimization, the average of fiber strain at BE and EE was 0.025 +/-0.011 (mean+/-standard deviation) and the difference in fiber strain during ejection was 0.214+/-0.018. The results indicate that the LV structure is designed for maximum homogeneity of fiber strain during ejection.

Genetic identification of Candida species in HIV-positive patients using the polymerase chain reaction and restriction fragment length polymorphism analysis of its DNA.

The polymerase chain reaction was used to amplify a targeted region: an internal transcribed spacer region of the ribosomal DNA from 114 Candida isolates and 65 reference strains. Unique product sizes were obtained for Candida glabrata, C. guillermondii and C. inconspicua. Isolates of C. albicans, C. tropicalis, C. dubliniensis and C. krusei could be identified following restriction digestion of the PCR products. The methods proved to be both simple and reproducible and may offer potential advantages over phenotyping methods.

Cross-sectional study of oral Candida carriage in a human immunodeficiency virus (HIV)-seropositive population: predisposing factors, epidemiology and antifungal susceptibility.

The Candida species isolated from oral rinses of 130 human immunodeficiency virus (HIV) infected patients were compared with those of 130 healthy non-matched volunteers. The oral rinses were plated on CHROMagar Candida medium (CAC) and on CAC supplemented with 10 micrograms (CF10) and 100 micrograms (CF100) of fluconazole per ml. The prevalence of non-albicans Candida spp. in oral rinses of HIV-infected patients and their correlation with the clinical and epidemiological characteristics of the patients were studied. Susceptibility of the Candida spp. isolated was determined by a microbroth dilution method based on the NCCLS reference procedure. Results of susceptibility tests of the yeast isolates were compared with their growth at the time of isolation on CAC supplemented with fluconazole. Thirty-five (30.7%) strains of non-albicans Candida spp. were isolated from the HIV-positive population, vs. seven (15.9%) from the immunocompetent population. Growth on CF10 correlated in 96% of the cases with fluconazole minimum inhibitory concentration (MIC) > 8 micrograms ml-1. Smoking and use of azoles were significantly associated with oral carriage of non-albicans Candida spp. (P < 0.05). The prevalence of non-albicans Candida spp. in HIV-positive persons in oral rinse samples is twice as high as in the HIV-negative population. Smoking and treatment with azoles are risk factors for the oral carriage of non-albicans Candida spp. The isolation of yeasts on CAC plates supplemented with fluconazole allows combination of presumptive yeast identification and fluconazole susceptibility testing.

Growth inhibition of human in vitro and mouse in vitro and in vivo mammary tumor models by retinoids in comparison with tamoxifen and the RU-486 anti-progestagen.

Retinoids constitute a very promising class of agents for the chemoprevention or treatment of breast cancer. These retinoids exert their biological activity through two distinct classes of retinoic acid (RA) receptors (R), the RAR isotypes (alpha, beta, and gamma) and the three RXR isotypes (alpha, beta, and gamma) and their numerous isoforms which bind as RXR/RAR heterodimers to the polymorphic cis-acting response elements of RA target genes. With respect to these numerous receptor sub-types, the retinoid-induced effects at the biological level include marked modifications with respect to both cell proliferation and cell death (apoptosis), and also in the induction of differentiation processes. The present study aims to characterize the effect which four retinoids (TTNPB, 9-cis-RA, LGD 1069, 4-HPR) with distinct RAR/RXR binding properties induced on various in vitro and in vivo mouse and human breast cancer models. The experiments with the retinoids were carried out in comparison with the anti-estrogen tamoxifen and the anti-progestagen RU-486 compounds. The results show that the 6 compounds under study were markedly more efficient in terms of growth inhibition in the human T-47D cell line when maintained under anchorage-independent culture conditions than when maintained under anchorage-dependent ones. While RU-486 exhibited a weak statistically significant (p < 0.05) influence on the growth of the T-47D stem cells, tamoxifen had a marked inhibitory influence on the growth of these cells. Of the four retinoids, 4-HPR was the least effective since the lowest doses tested (1 and 0.1 nM) exhibited no statistically (p > 0.05) significant influence on the growth of the stem cells. The most efficient retinoid was TTNPB. It was only at the highest dose (10 microM) that tamoxifen and RU-486 showed a weak inhibitory influence on the growth of the T-47D non-stem cells while all 4 retinoids exerted a significant inhibitory influence on the growth of these non-stem cells, with 4-HPR being the most efficient (P < 0.001) at the highest dose, but ineffective (P > 0.05) at the lowest. Tamoxifen and TTNPB were tested in vivo on hormone-sensitive (HS) and hormone-insensitive (HI) strains of the MXT murine mammary carcinoma. While TTNPB appeared to be equally efficient in terms of growth inhibition in both MXT-HS and MXT-HI models, tamoxifen had only a marginal inhibitory influence on the growth of the MXT-HI strain but did inhibit growth in the case of the MXT-HS one. TTNPB was markedly more efficient than tamoxifen in terms of both inhibiting the cell proliferation level (measured by means of computer-assisted microscopy applied to Feulgen-stained nuclei, a method which enables the percentage of cells in the S phase of the cell cycle to be determined) and triggering cell death (measured by means of the determination of the transglutaminase activity) in both the MXT-HI and MXT-HS models. The very significant TTNPB-induced inhibition of the macroscopic MXT-HS growth rate relates to the triggering of cell death (apoptosis) rather than to an inhibition of cell proliferation. All these results clearly indicate that retinoids are very efficient agents against breast cancer, at least as efficient as tamoxifen.

Optimization of cardiac fiber orientation for homogeneous fiber strain at beginning of ejection.

Mathematical models of left ventricular (LV) wall mechanics show that fiber stress depends heavily on the choice of muscle fiber orientation in the wall. This finding brought us to the hypothesis that fiber orientation may be such that mechanical load in the wall is homogeneous. Aim of this study was to use the hypothesis to compute a distribution of fiber orientation within the wall. In a finite element model of LV wall mechanics, fiber stresses and strains were calculated at beginning of ejection (BE). Local fiber orientation was quantified by helix (HA) and transverse (TA) fiber angles using a coordinate system with local r-, c-, and l-directions perpendicular to the wall, along the circumference and along the meridian, respectively. The angle between the c-direction and the projection of the fiber direction on the cl-plane (HA) varied linearly with transmural position in the wall. The angle between the c-direction and the projection of the fiber direction on the cr-plane (TA) was zero at the epicardial and endocardial surfaces. Midwall TA increased with distance from the equator. Fiber orientation was optimized so that fiber strains at BE were as homogeneous as possible. By optimization with TA = 0 degree, HA was found to vary from 81.0 degrees at the endocardium to -35.8 degrees at the epicardium. Inclusion of TA in the optimization changed these angles to respectively 90.1 degrees and -48.2 degrees while maximum TA was 15.3 degrees. Then the standard deviation of fiber strain (epsilon f) at BE decreased from +/- 12.5% of mean epsilon f to +/- 9.5%. The root mean square (RMS) difference between computed HA and experimental data reported in literature was 15.0 degrees compared to an RMS difference of 11.6 degrees for a linear regression line through the latter data.

Isolation of Candida species on media with and without added fluconazole reveals high variability in relative growth susceptibility phenotypes.

Mouthwashes from human immunodeficiency virus-positive individuals were sampled for yeasts by direct plating on a differential agar medium with and without added fluconazole and via enrichment broths with and without added fluconazole. The colonies of the yeasts isolated were tested for relative growth in the presence of single concentrations of itraconazole and fluconazole. Among 258 culture plates containing yeasts obtained via different isolation routes from 86 yeast-positive samples, 33 (12.7%) of the plates showed unexpectedly high colony-to-colony variation in relative growth. Intercolony variation was seen in 41 (47.7%) of the 86 isolates when relative growth data were analyzed for all colonies of an isolate tested, regardless of the medium used for isolation. The prevalence of relative growth variability with the azoles was highest for Candida glabrata (100% of 13 isolates), followed by Candida krusei (60% of 5 isolates) and Candida albicans (40% of 53 isolates), and the visual patterns of variability seen in scatter plots of the data showed species specificity. Relative growth phenotypes generally tended to be stable for each yeast colony in subcultures, whether or not the medium used for subculture contained antifungal agents. DNA fingerprinting of stable and variable C. albicans isolates showed changes in band patterns detected with the probe Ca3, suggesting that the variability may have resulted from selection of different subtypes of the yeasts during the isolation procedure. These findings suggest that the yeasts isolated from single clinical samples were often not clonal in nature. The relative growth test revealed colony variability more readily than conventional susceptibility testing.

A complementarity-determining region peptide of an anti-desmosome autoantibody may interact with the desmosomal plaque through molecular mimicry with a cytoplasmic desmoglein 1 sequence.

The sera of patients with pemphigus, a group of autoimmune blistering skin diseases, contain autoantibodies directed against components of adhering junctions termed desmosomes. F12, a human monoclonal antibody derived from a pemphigus patient, recognizes an unknown polypeptide of the desmosomal and hemidesmosomal plaques. The third complementarity-determining region of the F12 heavy chain (VH-CDR3) was shown to share a four-amino-acid sequence (GSSG) with the intracellular domains of desmoglein 1 and bullous pemphigoid antigen 2 which interact with components of, respectively, the desmosomal and hemidesmosomal plaques. Computer modeling of F12 showed that the GSSG sequence protudes inside the antigen-combining site and thus might be involved in antigen interactions. The GSSG sequence is essential to F12 function, since a peptide containing the VH-CDR3 inhibited its binding to target antigens while VH-CDR3 peptides with specific modifications of the GSSG sequence did not. These data allow us to hypothesize that certain autoantibodies produced during the course of an autoimmune disease can behave as adhesion molecules, through the molecular mimicry of the motif involved in protein/protein adhesion, and to propose a new self-antigen binding mechanism for some autoantibodies.

Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients.

During a study of oral rinses of 130 HIV-infected individuals, both typical and atypical Candida albicans colonies were isolated from ten patients on a yeast differential medium. Typical Candida albicans colonies were light green; atypical colonies were dark green. Both types of colonies were germ tube-positive and produced chlamydospores. However, DNA fingerprinting of the atypical isolates with the Ca3 Candida albicans-specific probe showed that they belonged to the recently described species Candida dubliniensis. Candida dubliniensis colonies could also be differentiated from Candida albicans colonies on isolation plates by the absence of fluorescence of colonies on methyl blue-Sabouraud agar under Wood's light. Among other phenotypic characteristics, only the absence of intracellular beta-glucosidase activity reliably distinguished Candida albicans from Candida dubliniensis. Candida dubliniensis may be underreported in clinical samples because most currently used isolation and identification methods fail to recognize this yeast.

Chemical synthesis, structural and functional characterisation of noxiustoxin, a powerful blocker of lymphocyte voltage-dependent K+ channels.

Two forms of the Centrudoides noxius scorpion noxiustoxin, containing an amidated and an acid C-terminus, were synthesized on a solid support by using Fmoc-chemistry and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling. Comparison of the two synthetic forms with the native toxin by tryptic mapping and CD spectroscopy shows that noxiustoxin possesses an amidated C-terminus and the same fold as all short scorpion toxins. Patch-clamp assays on B lymphocytes demonstrate that noxiustoxin inhibits the voltage-dependent K+ channels with 2 nM affinity, but does not affect the Ca(2+)-activated K+ channels. This toxin, because of its high affinity and specificity for voltage-gated K+ channel, may provide a powerful tool in the investigation of the role(s) of these channels in the T and B lymphocyte activation and proliferation.