Asymmetric alkene epoxidation by Mn(III)salen catalyst in ionic liquids

The enantioselective epoxidation of 6-cyano-2,2-dimethylchromene (Chrom) catalysed by the Jacobsen catalyst, using sodium hypochlorite (NaOCl) as oxygen source, at room temperature, was performed in a series of 1,3-dialkylimidazolium and tetra-alkyl-dimethylguanidium based ionic liquids. All the room temperature ionic liquids (RTILs) could be used as reaction media for the enantioselective epoxidation of the alkene giving, generally, moderate to good epoxide yields and enantiomeric excesses (ee%).For the series of ionic liquids derived from the 1,3-dialkylimidazolium cation, it was observed some relationship between the RTILs physical properties and the catalytic reaction parameters, exemplified by linear correlations between (i) the ee% and the α Kamlet-Taft parameter (hydrogen bond acidity of the solvent) for CH₂Cl₂ and [C₄m_nim][BF₄] ionic liquids (n = 1 or 2), and (ii) the ee% and the β Kamlet-Taft parameter (hydrogen bond basicity of the solvent) for CH₂Cl₂ and [C₄mim][X] ionic liquids (X = PF₆, NTf₂ or BF₄).All the RTILs could be reused in further catalytic cycles, with the exception of [C₈mim][PF₆]. The reutilisation of the Jacobsen catalyst for four times generally led to a decrease in the epoxide yield and to a slight decrease in the enantioselectivity. The recycling of the catalyst could be improved by imparting an ionic character to the complex through abstraction of the axially coordinated chloride anion (Cat 2). Other oxygen sources, such as iodosylbenzene, hydrogen peroxide and urea-hydrogen peroxide adduct, were also tested coupled with Jacobsen catalyst, but the best results were achieved with NaOCl.     

Isolation and characterisation of the biological repeating unit of cepacian, the exopolysaccharide produced by bacteria of the Burkholderia cepacia complex

The repeating unit of cepacian, the exopolysaccharide produced by the majority of the microorganisms belonging to the Burkholderia cepacia complex, was isolated from inner bacterial membranes and investigated by mass spectrometry, with and without prior derivatisation. Interpretation of the mass spectra led to the determination of the biological repeating unit primary structure, thus disclosing the nature of the oligosaccharide produced in vivo. Moreover, mass spectra recorded on the native sample revealed that acetyl substitution was very variable, producing a mixture of repeating units containing zero to four acyl groups. At the same time, finding acetylated oligosaccharides showed that binding of these substituents occurred in the cellular periplasmic space, before the polymerisation process took place. In the chromatographic peak containing the repeating unit, oligosaccharides shorter than the repeating unit co-eluted. Mass spectrometric analysis showed that they were biosynthetic intermediates of the repeating unit and further investigation revealed the biosynthetic sequence of cepacian building block.     

TLR4 recognizes Pseudallescheria boydii conidia and purified rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.     

Autonomic regulation of pacemaker activity: role of heart nitric oxide synthases

In autonomic-blocked rats treated with NG-nitro-L-arginine methyl ester (L-NAME, 7.5 mg/kg), heart rate increased 18% and mean arterial pressure increased 48%. Thyroidectomy, along with autonomic blockade, hampered the chronotropic response but did not modify the effect on blood pressure. After 150 min of autonomic blockade, the experimental end point, total nitric oxide (NO) production by heart NO synthases (NOS) decreased 61%: from 54 to 21 nmol NO.min-1.g heart-1. Mitochondrial NOS (mtNOS) and sarcoplasmic reticulum endothelial NOS activities decreased 74% and 52%, respectively. Mitochondria isolated from whole heart showed a well-coupled oxidative phosphorylation with high respiratory control and ADP-to-O ratios, decreased mtNOS activity (55-60%), and decreased mtNOS protein expression (70%). Immunohistochemistry with anti-inducible NOS antibody linked to gold particles localized mtNOS at the inner mitochondrial membranes. Histochemical right atrial NOS (NADPH-diaphorase) decreased 55% after heart denervation. The effects of autonomic denervation on the NO system were partially prevented by thyroidectomy performed simultaneously with autonomic blockade. Western blot analysis indicated a very rapid mtNOS protein turnover (half time=120 min) with a process of protein expression that was upregulated by thyroidectomy and a degradation process that was downregulated by the autonomic nervous system. The observations suggest that NO-mediated pathways contribute to pacemaker heart activity, likely through the NO steady-state levels in the right atrium and the whole heart.     

Role of nitric oxide and prostanoids in attenuation of rapid baroreceptor resetting

Because the regulation of vascular function involves complex mutual interactions between nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) products, we examined the contribution of NO and prostanoids derived from the COX pathway in modulating aortic baroreceptor resetting during an acute (30 min) increase in arterial pressure in anesthetized rats. Increase in pressure was induced either by administration of the nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or aortic coarctation (COA) with or without treatment with the COX inhibitor indomethacin (INDO) or the selective neuronal NOS inhibitor 1-(2-trifluoromethylphenyl)imidazole (TRIM). The activity of the aortic depressor nerve and arterial pressure were simultaneously recorded, and the degree of resetting was determined by the shift of the pressure-nerve activity curve using the ratio [delta systolic pressure at 50% of maximum baroreceptor activity/delta systolic pressure] x 100. The magnitude of pressure rise was similar in the different groups (59 +/- 6, 53 +/- 5, 53 +/- 5, 45 +/- 5, 49 +/- 3, and 41 +/- 3 mmHg for COA, L-NAME, INDO+COA, INDO+L-NAME, TRIM+COA, and TRIM+INDO+COA, respectively, P = 0.27). The degree of resetting that occurred with L-NAME or COA combined with treatment with TRIM was attenuated compared with COA alone (7 +/- 4, 5 +/- 2, and 31 +/- 6%, respectively, P = 0.04). INDO failed to influence baroreceptor resetting to higher pressure but prevented L-NAME- and TRIM-induced effects (20 +/- 7, 21 +/- 8, and 32 +/- 6% for INDO+COA, INDO+L-NAME, and INDO+TRIM+COA, respectively; P = 0.38). Baroreceptor gain was affected only by l-NAME. These findings indicate that NO, probably from neuronal origin, may exert stimulatory influence on the degree of rapid baroreceptor resetting to hypertension that involves COX-derived prostanoids.     

Adjuvant effect of the Propionibacterium acnes and its purified soluble polysaccharide on the immunization with plasmidial DNA containing a Trypanosoma cruzi gene

In the present work we investigated the role of killed Propionibacterium acnes or a soluble polysaccharide extracted from bacterium cell wall in modulated experimental immunization with plasmidial DNA. We used a plasmid, p154/13, containing a gene-encoding catalytic domain of Trypanosoma cruzi (T. cruzi) trans-sialidase. As previously described, immunization of BALB/c mice with p154/13 elicited humoral, cell-mediated and protective immune responses against T. cruzi infection. In this study we describe that both P. acnes and its soluble polysaccharide fraction have the ability to modulate the immune response elicited by p154/13. Treatment with these adjuvants enhanced specific trans-sialidase Th1 immune response, as revealed by a lower IgG1/IgG2a ratio and stronger in vitro IFN-gamma synthesis by CD4+ T cells. The most important fact was that treatment with P. acnes or its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzi challenge. These data suggest that P. acnes or its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruzi infection.     

A computational protocol to probe the role of solvation effects on the reduction potential of azurin mutants

Semiquantitative relationships between thermodynamic parameters of Cu2+ reduction experimentally measured for a series of azurin mutants and the solvation free energy of the oxidized state of the proteins were derived. Solvation free energy calculations were carried out within an ONIOM/PCM scheme specifically adapted to this protein series. The method proved to be able to capture the main determinants of the measured reduction parameters, providing satisfactory predictions of the E degrees '.     

Antiproliferative effects of tocopherols (vitamin E) on murine glioma C6 cells: homologue-specific control of PKC/ERK and cyclin signaling

Chemoprevention strategies for brain tumors (specifically gliomas) are few and surprisingly poorly investigated. We have studied the effects of tocopherols (TOCs; vitamin E) on proliferation and death processes of murine glioma C6 cells. These vitamers showed different cell uptake and concentration- and time-dependent inhibitory effects on cell growth that were significant at the lowest concentrations tested (1-10 microM). However, the inhibitory potency of TOCs seemed to reflect at least in part their actual cell concentrations at steady state, with the order of magnitude gamma-TOC >or= alpha-TOC > delta-TOC approximately or = beta-TOC. Moreover, for extracellular concentrations >or=10 microM, TOCs also showed a significant cytotoxic effects due mainly to necrosis, while apoptosis was negligible. Gamma-TOC (the form showing preferential cell uptake and lowest unspecific cytotoxicity) was the most effective inhibitor of cell cycle progression (arrest in G0/G1 phase) leading to lowered expression of cyclin E and cyclin-dependent kinases 2 and 4 and overexpression of p27 (specific inhibitor of S-phase entering). According to these signals, activated ERK1/2 and PKC upstream and Rb phosphorylation downstream were decreased. In conclusion, within TOCs the gamma form exerts the most potent and specific control of cell cycle progression in C6 cells (cytostatic effect). This suggests a chemopreventive role of this form of vitamin E in gliomas.