Non-induced leukocyte extract reduces HIV replication and TNF secretion

According to UNAIDS, the global HIV/AIDS epidemic increased to 40 million the number of people living with the virus around the world. Dialyzable leukocyte extract obtained by our group is a low molecular weight dialyzable material from peripheral human leukocytes previously in vitro induced with Sendai virus (DLE-ind), and more recently, from non-induced leukocytes (DLE n/i). Previous results have shown the ability of DLE-ind to inhibit HIV in vitro replication in MT4 cell; to reduce TNFalpha secretion, and to delay in vivo progression to AIDS in early stage of HIV infection. In this work we present evidences that DLE n/i also inhibits HIV in vitro replication and reduces TNFalpha secretion in human whole blood like DLE obtained from induced leukocytes. Taking together these results show that both properties of DLE, HIV in vitro inhibition and TNF production modulation, are not dependent on in vitro Sendai virus induction of leukocytes.     

Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E(#) with monodisperse diheptanoylphosphatidylcholine (DC(7)PC) that is distinguishable from the E complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC(7)PC give an apparent dissociation constant of K(2) = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E(#) are distinct from those of the E complex. The aggregation and partitioning behaviors suggest that the acyl chains in E(#) but not in E remain exposed to the aqueous phase. The free (E) and complexed (E(#) and E) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC(7)PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis-Menten complex formed with monodisperse DC(7)PC is likely to be E(#)S or its aggregate rather than the classical monodisperse ES complex.     

Characterization of liposomal tacrolimus in lung surfactant-like phospholipids and evaluation of its immunosuppressive activity

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent that rapidly penetrates the plasmatic membrane and inhibits the signal transduction cascade of T lymphocytes. The objective of this study was the characterization of liposomal FK506 with surfactant-like phospholipids to be administered intratracheally after lung transplantation or in inflammatory lung diseases. We evaluated the optimal incorporation of FK506 in dipalmitoylphosphatidylcholine (DPPC) and DPPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) monolayers and bilayers and the effects of FK506 on the physical properties of DPPC and DPPC/POPG (8:2 w/w) vesicles. In addition, we assessed the immunosuppressive effects of surfactant-like phospholipid vesicles containing different amounts of FK506 on T-cell proliferation and interleukin 2 production. From surface pressure measurements of FK506/DPPC and FK506/DPPC/POPG mixed monolayers, we determined that FK506 was embedded into these monolayers up to an FK506 concentration of about 0.4 mol %. Beyond this concentration, FK506 was not quantitatively incorporated into the monolayer, suggesting possible concentration-dependent aggregation of tacrolimus. The incorporation of FK506 into DPPC monolayers, at concentrations 相似文献   

Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and .NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker beta-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the .NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization.     

Early population differentiation in extinct aborigines from Tierra del Fuego-Patagonia: ancient mtDNA sequences and Y-chromosome STR characterization

Ancient mtDNA was successfully recovered from 24 skeletal samples of a total of 60 ancient individuals from Patagonia-Tierra del Fuego, dated to 100-400 years BP, for which consistent amplifications and two-strand sequences were obtained. Y-chromosome STRs (DYS434, DYS437, DYS439, DYS393, DYS391, DYS390, DYS19, DYS389I, DYS389II, and DYS388) and the biallelic system DYS199 were also amplified, Y-STR alleles could be characterized in nine cases, with an average of 4.1 loci per sample correctly typed. In two samples of the same ethnic group (Aonikenk), an identical and complete eight-loci haplotype was recovered. The DYS199 biallelic system was used as a control of contamination by modern DNA and, along with DYS19, as a marker of American origin. The analysis of both mtDNA and Y-STRs revealed DNA from Amerindian ancestry. The observed polymorphisms are consistent with the hypothesis that the ancient Fuegians are close to populations from south-central Chile and Argentina, but their high nucleotide diversity and the frequency of single lineages strongly support early genetic differentiation of the Fuegians through combined processes of population bottleneck, isolation, and/or migration, followed by strong genetic drift. This suggests an early genetic diversification of the Fuegians right after their arrival at the southernmost extreme of South America.     

In vivo two-photon laser-scanning microscopy of Ca2+ dynamics in visual motion-sensitive neurons

We applied two-photon laser-scanning microscopy (TPLSM) to motion-sensitive visual interneurons of the fly to study Ca(2+) dynamics in vivo at a higher spatial and temporal resolution than possible with conventional fluorescence microscopy. Based on a custom-built two-photon microscope, we performed line scans to measure changes in presynaptic Ca(2+) concentrations elicited by visual stimulation. We used a fast avalanche photodiode (APD) with a high quantum efficiency to detect even low levels of emitted fluorescence. Our experiments show that our in vivo preparation is amenable to TPLSM: with excitation intensities low enough not to cause photodamage, activity-dependent fluorescence changes of Ca(2+)-sensitive dyes can be detected in small neuronal branches. The performance of two-photon and conventional Ca(2+) imaging carried out consecutively at the same neuron is compared and it is demonstrated that two-photon imaging allows us to detect differences in Ca(2+) dynamics between individual neurites.     

Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)相似文献   

Binding of xanthine oxidase to glycosaminoglycans limits inhibition by oxypurinol

Although the binding of xanthine oxidase (XO) to glycosaminoglycans (GAGs) results in significant alterations in its catalytic properties, the consequence of XO/GAG immobilization on interactions with clinically relevant inhibitors is unknown. Thus, the inhibition kinetics of oxypurinol for XO was determined using saturating concentrations of xanthine. When XO was bound to a prototypical GAG, heparin-Sepharose 6B (HS6B-XO), the rate of inactivation for uric acid formation from xanthine was less than that for XO in solution (k(inact) = 0.24 versus 0.39 min(-1)). Additionally, the overall inhibition constant (K(i)) of oxypurinol for HS6B-XO was 2-5-fold greater than for free XO (451 versus 85 nm). Univalent electron flux (O(2)(.) formation) was diminished by the binding of XO to heparin from 28.5% for free XO to 18.7% for GAG-immobilized XO. Similar to the results obtained with HS6B-XO, the binding of XO to bovine aortic endothelial cells rendered the enzyme resistant to inhibition by oxypurinol, achieving approximately 50% inhibition. These results reveal that GAG immobilization of XO in both HS6B and cell models substantially limits oxypurinol inhibition of XO, an event that has important relevance for the use of pyrazolo inhibitors of XO in clinical situations where XO and its products may play a pathogenic role.     

Evaluation of the European Union Maize Landrace Core Collection for resistance to Sesamia nonagrioides (Lepidoptera: Noctuidae) and Ostrinia nubilalis (Lepidoptera: Crambidae)

Two corn borer species are the principal maize insect pests in Europe, the European corn borer, Ostrinia nubilalis (Hübner), and the pink stem borer, Sesamia nonagrioides (Lefebvre). Hence, it would be advisable to evaluate the European maize germplasm for corn borer resistance to generate European varieties resistant to corn borer attack. The creation of the European Union Maize Landrace Core Collection (EUMLCC) allowed the screening of most of the variability for European corn borer resistance present among European maize local populations from France, Germany, Greece, Italy, Portugal, and Spain, testing a representative sample. The objective of this study was the evaluation of stem and ear resistance of the EUMLCC to European corn borer and pink stem borer attack. Trials were made at two Spanish locations that represent two very different maize-growing areas. Populations that performed relatively well under corn borer infestation for stem and ear damage were 'PRT0010008' and'GRC0010085', among very early landraces; 'PRT00100120' and 'PRT00100186', among early landraces; 'GRC0010174', among midseason landraces; and 'ESP0070441', among late landraces. Either the selection that could have happen under high insect pressure or the singular origin of determined maize populations would be possible explanations for the higher corn borer resistance of some landraces. Landraces 'PRT0010008', 'FRA0410090', 'PRT00100186', and 'ESP0090214' would be selected to constitute a composite population resistant to corn borers and adapted to short season, whereas populations 'ESP0090033', 'PRT00100530', 'GRC0010174', and 'ITA0370005' would be used to make a resistant composite adapted to longer season.     

Transient corticosteroid treatment permanently amplifies the Th2 response in a murine model of asthma

Corticosteroids (CS) remain the most efficacious pharmacotherapeutic option for the management of asthma. Although the acute anti-inflammatory effects of CS treatment have been amply documented both clinically and experimentally, recent human data intimate that exposure to CS may be associated with retrograde immune phenomena, including enhanced synthesis of IgE in vivo and elevated Th2 cytokine production in vitro. We have investigated the long-term immunologic effects of CS treatment in a murine model of allergic airway inflammation. CS treatment during initial exposure to OVA or upon long-term Ag rechallenge remarkably attenuated eosinophilic airway inflammation and airway hyperresponsiveness. Interestingly, however, Th2 cytokine production by cultured splenocytes from CS-treated mice was significantly elevated, while IFN-gamma synthesis was depressed. Moreover, mice rechallenged with OVA several weeks after CS intervention during allergic sensitization not only developed airway inflammation, but also exhibited enhanced Th2 cytokine production in lymphoid tissues and OVA-specific IgE in serum. This amplification of the systemic immune response was associated with an intact APC compartment during CS-conditioned sensitization to OVA. These data indicate that immune processes underlying the allergic phenotype remain impervious to CS treatment and raise the possibility that treatment with CS during sensitization may amplify elements of the allergen-specific immune response.