Directed evolution and targeted mutagenesis to murinize <Emphasis Type="Italic">listeria monocytogenes</Emphasis> internalin A for enhanced infectivity in the murine oral infection model

Background  

Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.     

Refolding of hexameric porcine leucine aminopeptidase using a cationic detergent and dextrin-10 as artificial chaperones

The application of artificial chaperones in biotechnology has been inspired by the mechanism of molecular chaperones like GroEL/GroES. It involves addition of a capturing detergent during dilution of the chaotropic reagent, that prevents protein aggregation, and finally, addition of a oligosaccharide that removes the detergent allowing the protein to refold. Here, guanidinium hydrochloride-denatured hexameric leucine aminopeptidase is shown to be efficiently refolded by using the cationic detergent cetyltrimethylammonium bromide and the linear polysaccharide dextrin-10 as artificial chaperones. The effect of these additives and the time dependence on the recovery of total enzymatic activity, kinetic parameters (K_M, k_cat), intrinsic steady-state tryptophan fluorescence and oligomeric structure is presented. The method described is very promising since 92% of fully active and correct folded LAP could be produced. Moreover, we showed that the stripping process is relatively slow, it allows the protein to refold almost entirely to its native state.     

High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus

Introduction  

High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity.     

Vasoactive intestinal peptide inhibits cytokine production in T lymphocytes through cAMP-dependent and cAMP-independent mechanisms.

Previous reports indicate that VIP and the structurally related peptide PACAP, inhibit IL-2 and IL-10 production in antigen-stimulated T lymphocytes. Intracellular cAMP elevation appears to be the primary transduction pathway involved. However, in the lower concentration range, an additional, cAMP-independent transduction pathway appears to mediate the VIP inhibition of cytokine production. Here, we address this question by using VIP agonists and antagonists which act through cAMP-dependent and -independent pathways. The antagonists based on the neurotensin-VIP hybrid molecule did not affect the inhibitory effect of VIP/PACAP on IL-2 and IL-10 production, confirming that astrocytes and T lymphocytes express different receptors. A lipophilic antagonist with increased membrane permeability, partially reversed the inhibitory effect of VIP/PACAP, forskolin, prostaglandin E2, and 8-bromo-cAMP without significantly affecting cAMP levels, suggesting that it acts downstream of cAMP. Two VIP agonists inhibit IL-2 and IL-10 production. One of the agonists increases cAMP, whereas the second one does not induce cAMP/cGMP. Our results indicate that VIP inhibits cytokine production in stimulated CD4+ T cells through two separate mechanisms, which involve both cAMP-dependent and cAMP-independent transduction pathways.