Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position

To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (≥0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity.     

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.     

Broad-spectrum antioxidant peptides derived from His residue-containing sequences present in human paraoxonase 1

Hydroxyl or peroxyl radicals and hypochlorous acid (HOCl) are known to cause the oxidation of lipoproteins. Here, we examined Cu²⁺-binding property of paraoxonase 1 (PON1), and antioxidant actions of peptides, resembling His residue-containing sequences in PON1, against oxidations by Cu²⁺, peroxyl radicals or HOCl. When Cu²⁺-binding property of PON1 was examined spectrophotometrically, the maximal Cu²⁺ binding was achieved at 1:1 molar ratio of PON1: Cu²⁺. Additionally, Cu²⁺-catalyzed oxidative inactivation of PON1 was prevented by Ca²⁺-depleted PON1 at 1:1 ratio, but not diethylpyrocarbonate (DEPC)-modified PON1, suggesting the participation of His residue in Cu²⁺-binding. When His-containing peptides were examined for antioxidant actions, those with either His residue at N-terminal position 2 or 3, or His-Pro sequence at C-terminal remarkably prevented Cu²⁺-mediated low density lipoprotein (LDL) oxidation and PON1 inactivation. Especially, FHKALY, FHKY or NHP efficiently prevented Cu²⁺-induced LDL oxidation (24 h), indicating a tight binding of Cu²⁺ by peptides. In support of this, the peptide/Cu²⁺ complexes exhibited a superoxide-scavenging activity. Separately, in oxidations by 2,2'-azobis-2-amidinopropane hydrochloride or HOCl, the presence of Tyrosine (Tyr) or Cysteine (Cys) residue markedly enhanced antioxidant action of His-containing peptides. These results indicate that His-containing peptides with Tys or Cys residues correspond to broad spectrum antioxidants in oxidation models employing Cu²⁺, 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) or HOCl.     

In silico pharmacogenetics of warfarin metabolism

Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.     

Thermodynamic confinement and alpha-helix persistence length in poly(gamma-benzyl-L-glutamate)-b-poly(dimethyl siloxane)-b-poly(gamma-benzyl-L-glutamate) triblock copolymers

The structure and the associated dynamics of a series of poly(gamma-benzyl-L-glutamate)-b-poly(dimethyl siloxane)-b-poly(gamma-benzyl-L-glutamate) (PBLG-b-PDMS-b-PBLG) triblock copolymers were investigated using small- and wide-angle X-ray scattering, NMR, transmission electron microscopy, and dielectric spectroscopy, respectively. The structural analysis revealed phase separation in the case of the longer blocks with defected alpha-helical segments embedded within the block copolymer nanodomains. The alpha-helical persistence length was found to depend on the degree of segregation; thermodynamic confinement and chain stretching results in the partial annihilation of helical defects.     

Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.     

Gene expression profiling of breast carcinomas using nylon DNA arrays

Clinically very heterogeneous, breast cancer prognosis and treatment response are difficult to predict with the current prognostic histoclinical parameters. Mammary oncogenesis remains poorly understood. DNA array technology allows the simultaneous analysis of the mRNA expression levels of thousands of genes in biological samples. Applied to breast tumours, expression profiles will boost our knowledge of oncogenesis, will offer new potential therapeutic targets and new prognostic and predictive markers. Today, the most accessible approach for academic research teams is that of Nylon DNA arrays with radioactive detection, which in addition allows profiling of small clinical samples.