Novel S-acyl glutathione derivatives prevent amyloid oxidative stress and cholinergic dysfunction in Alzheimer disease models

Oxidative stress-mediated neuronal death may be initiated by a decrease in glutathione (GSH), whose levels are reduced in mitochondrial and synaptosomal fractions of specific CNS regions in Alzheimer disease (AD) patients. Currently, the use of GSH as a therapeutic agent is limited by its unfavorable pharmacokinetic properties. In this study, we designed the synthesis of new S-acyl glutathione (acyl-SG) thioesters of fatty acids via N-acyl benzotriazole-intermediate production and investigated their potential for targeted delivery of the parent GSH and free fatty acid to amyloid-exposed fibroblasts from familial AD patients and human SH-SY5Y neuroblastoma cells. Cell culture supplementation with acyl-SG derivatives triggers a significant decrease in lipid peroxidation and mitochondrial dysfunction in a fatty acid unsaturation degree-dependent fashion. Acyl-SG thioesters also protect cholinergic neurons against Aβ-induced damage and reduce glial reaction in rat brains. Collectively, these findings suggest that acyl-SG thioesters could prove useful as a tool for controlling AD-induced cerebral deterioration.     

Human CD25+ regulatory T cells maintain immune tolerance to nickel in healthy, nonallergic individuals

We investigated the capacity of CD25(+) T regulatory cells (Treg) to modulate T cell responses to nickel, a common cause of allergic contact dermatitis. CD4(+) T cells isolated from the peripheral blood of six healthy, nonallergic individuals showed a limited capacity to proliferate in response to nickel in vitro, but responsiveness was strongly augmented (mean increment +/- SD, 240 +/- 60%) when cells were depleted of CD25(+) Treg. Although CD25(+) Treg were anergic to nickel, a small percentage up-regulated membrane CTLA-4 upon nickel exposure. CD25(+) Treg strongly and dose-dependently inhibited nickel-specific activation of CD25(-) T lymphocytes in coculture experiments in a cytokine-independent, but cell-to-cell contact-dependent, manner. Approximately 30% of circulating CD25(+) Treg expressed the cutaneous lymphocyte-associated Ag (CLA), and CLA(+)CD25(+) Treg were more efficient than CLA(-)CD25(+) cells in suppressing nickel responsiveness of CD25(-) T cells. The site of a negative patch test in response to nickel showed an infiltrate of CD4(+)CLA(+) cells and CD25(+) cells, which accounted for approximately 20% of the total T cells isolated from the tissue. Skin-derived T cells suppressed nickel-specific responses of peripheral blood CD25(-) T cells. In addition, 60 +/- 14% of peripheral blood CD25(+) Treg expressed the chemokine receptor CCR7 and strongly inhibited naive T cell activation in response to nickel. Finally, CD25(+) T cells isolated from peripheral blood of nickel-allergic patients showed a limited or absent capacity to suppress metal-specific CD4(+) and CD8(+) T cell responses. The results indicates that in healthy individuals CD25(+) Treg can control the activation of both naive and effector nickel-specific T cells.     

Relationships of ligand binding,redox properties,and protonation in Coprinus cinereus peroxidase

The pH dependence of the redox potentials and kinetics for CO association and dissociation was determined between pH 3.0 and 13.0 at 25 degrees C for the wild-type Coprinus cinereus fungal peroxidase and for a site-directed mutant in which Asp245, which is H-bonded to N delta of the imidazole of the proximal His183, was substituted with Asn. The determination of these functional properties allowed this information to be merged in a self-consistent fashion and to formulate for the first time a complete scheme employing the minimum number of groups required to describe the whole proton-linked behavior of both redox and ligand binding properties. The overall pH dependence can be accounted for by four redox- and ligand-linked groups. The proximal H-bond, which is strictly conserved in all peroxidases, will still be present in the site-specific mutant, but will no longer have an ionic character, and this event will bring about an alteration of redox equilibria and CO binding kinetics, envisaging a relevant role played by this H-bond also in modulating redox properties and ligand binding equilibria.     

Leitbakteria of microbial biofilm communities causing occlusion of biliary stents

Biliary stents inserted to relieve obstructive jaundice caused by biliary or pancreatic malignancies inevitably become occluded by microbial growth in the form of diverse microbial community biofilms. The scarce information available on these communities is based on cultivation methods, but such methods usually provide distorted overviews of community composition, so commonalities and differences in biliary stent communities are uncertain. We extracted DNA and RNA from the microbial communities of 11 biliary stents explanted from nine patients in hospitals from two different countries, amplified 16S rRNA and rDNA sequences, analysed the amplicons by the single-strand conformation polymorphism (SSCP) method, and sequenced and deduced phylogenetic assignments of the major amplicons representing the major biofilm community members. We used a Modified Robbins Device (MRD) to study de novo development of a stent biofilm from a patient stent microbial community. Single-strand conformation polymorphism fingerprinting revealed the same six abundant bacterial species, here designated Leitbakteria, namely Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter aerogenes, and two unculturable bacteria distantly related to E. coli and Shigella sonnei, in all of the stent biofilm communities. In the experimental biliary stent system, a sequential colonization of the stent surface was observed, with P. aeruginosa being the pioneer colonizer, followed by K. pneumoniae and one of the unculturable Leitbakteria, followed by the remainder of the community. The overview of microbial biofilm communities of biliary stents gained by the use of culture-independent methods revealed new unculturable bacteria as major members of biliary stent biofilms, and the diversity of the abundant members of the stent biofilms is considerably lower than suggested from earlier studies based on cultivation methods, and that communities from different stents from different patients in different countries are remarkably similar and have similar major members, the stent Leitbakteria.     

Methyltrioxorhenium catalysed synthesis of highly oxidised aryltetralin lignans with anti-topoisomerase II and apoptogenic activities

A novel and efficient procedure to prepare highly oxidised aryltetralin lignans, such as isopodophyllotoxone and (-)-aristologone derivatives, by oxidation of podophyllotoxin and galbulin with methylrhenium trioxide (MTO) and novel MTO heterogeneous catalysts is reported. It is noteworthy that in the case of isopodophyllotoxone derivatives the functionalisation of the C-4 position of the C-ring and the ring-opening of the D-lactone moiety increased the activity against topoisomerase II while causing the undesired inhibition of tubulin polymerisation to disappear. The novel (-)-aristologone derivatives showed apoptogenic activity against resistant human lymphoma cell lines.     

Human cytomegalovirus proteins pp65 and immediate early protein 1 are common targets for CD8+ T cell responses in children with congenital or postnatal human cytomegalovirus infection

Recombinant modified vaccinia Ankara- and peptide-based IFN-gamma ELISPOT assays were used to detect and measure human CMV (HCMV)-specific CD8(+) T cell responses to the pp65 (UL83) and immediate early protein 1 (IE1; UL123) gene products in 16 HCMV-infected infants and children. Age at study ranged from birth to 2 years. HCMV-specific CD8(+) T cells were detected in 14 (88%) of 16 children at frequencies ranging from 60 to >2000 spots/million PBMC. Responses were detected as early as 1 day of age in infants with documented congenital infection. Nine children responded to both pp65 and IE1, whereas responses to pp65 or IE1 alone were detected in three and two children, respectively. Regardless of the specificity of initial responses, IE1-specific responses predominated by 1 year of age. Changes in HCMV epitopes targeted by the CD8(+) T cell responses were observed over time; epitopes commonly recognized by HLA-A2(+) adults with latent HCMV infection did not fully account for responses detected in early childhood. Finally, the detection of HCMV-specific CD8(+) T cell responses was temporally associated with a decrease in peripheral blood HCMV load. Taken altogether, these data demonstrate that the fetus and young infant can generate virus-specific CD8(+) T cell responses. Changes observed in the protein and epitope-specificity of HCMV-specific CD8(+) T cells over time are consistent with those observed after other primary viral infections. The temporal association between the detection of HCMV-specific CD8(+) T cell responses and the reduction in blood HCMV load supports the importance of CD8(+) T cells in controlling primary HCMV viremia.     

Role of interleukin 2 (IL 2) and hemopoietin-1 (H-1) in the generation of mouse natural killer (NK) cells from primitive bone marrow precursors

The development of natural killer (NK) cells from bone marrow (BM) precursors was studied. Recombinant interleukin 2 (IL 2) was able to induce the in vitro development of NK cells when added to cultures of mouse BM cells. Treatment of donor mice with 5-fluorouracil (150 mg/kg i.v.), which eliminates more differentiated cells but spares less differentiated cells, appears to augment NK cell development. The "NK stem cell" was found to be asialo GM1-, Thy-1+, Lyt-2-, and Lyt-1-. The cells generated in vitro had a typical phenotype of NK cells, being asialo GM1+, Lyt-5+, Thy-1+, Lyt-2-, and Lyt-1-. These effector cells also had specificity characteristics of NK cells lysing the NK-susceptible YAC-1 and K562 targets, but not the NK-resistant EL/4 or allogeneic and syngeneic blasts. Hemopoietin-1 (H-1), a factor which acts on very primitive multipotent BM cells, was able to cooperate with IL 2, increasing the development of NK cells. In contrast, other factors such as interleukin 3 or colony-stimulating factor did not cause induction of NK activity when added to cultures of BM cells, indicating that this effect, i.e., induction of NK cell development, is peculiar to IL 2. These results indicate that IL 2 can act as a differentiation as well as growth factor for NK cells, and that H-1 can promote the development of functional activity in a lymphocyte subpopulation as well as affect the differentiation of myelomonocytic and other cell lineages. This experimental system appears quite useful for characterization of BM precursors for NK cells, and should help to better understand the relationship of the NK cell lineage to the T cell or other lineages.     

Dynamic fluorescence in copper proteins Selected examples

Summary The fluorescence properties of three copper proteins, namely human superoxide dismutase,Pseudomonas aeruginosa azurin andThiobacillus versutus amicyanin have been studied. All these proteins show a non-exponential decay of fluorescence, though the tryptophanyl residues responsible for the emission are very differently located in the three proteins. All the three decays can be fitted by at least two lifetimes or better with one or two lorentzian-shaped, continuous distributions of lifetime. In each case the removal of copper affects the quantum yield of fluorescence without affecting the shape of the emission.