Prostaglandin E2 inhibits TNF production in murine bone marrow-derived dendritic cells

Exposure to pathogens induces dendritic cells to release inflammatory cytokines and chemokines. The inflammatory response is controlled by endogenous agents such as anti-inflammatory cytokines, glucocorticoids, anti-inflammatory neuropeptides, and lipid mediators. This study is the first report on the inhibition by prostaglandin E2 (PGE2) of TNF release from bone marrow-derived dendritic cells stimulated with lipopolysaccharide (LPS), a TLR4 ligand, or peptidoglycan, a TLR2 ligand. The inhibition of TNF occurs at both mRNA and protein level. The inhibitory effect of PGE2 is mediated by the EP2 and EP4 receptors, and involves both PKA signaling and mediation by DC-derived IL-10. Intraperitoneal administration of PGE2 together with LPS results in a reduction in serum TNF and intracellular TNF in peritoneal exudate cells, compared to LPS alone. In addition, administration of PGE2 in vivo reduces the numbers of CD11c+ DCc that accumulate in the peritoneal cavity in response to LPS. The various implications of the PGE2-induced reduction in TNF are discussed.     

alpha-crystallin protects glucose 6-phosphate dehydrogenase against inactivation by malondialdehyde

The present work investigates the effect of malondialdehyde (MDA) binding on the enzymic activity and on some structural properties of glucose 6-phosphate dehydrogenase (G6PD). We studied whether alpha-crystallin could protect the enzyme against MDA damage, and if so, by what mechanism. We also studied whether alpha-crystallin could renature G6PD denatured by MDA. alpha-Crystallin was prepared from bovine lenses by gel chromatography. MDA was freshly prepared and incubated with G6PD with or without alpha-crystallin. The results show that MDA reacted with G6PD non-enzymically causing inactivation at concentrations lower than those used previously on structural proteins. The modified enzyme became fluorescent. alpha-Crystallin, acting as a molecular chaperone, specifically protected the enzyme against inactivation by MDA. The enzyme was not reactivated by alpha-crystallin, but it was stabilised and protected against further denaturation. Complex formation between alpha-crystallin and the modified enzyme was demonstrated by immunoprecipitation. G6PD was very susceptible to MDA and we have shown for the first time that alpha-crystallin is able to protect the enzyme against this damage.     

Granzyme B, a new player in activation-induced cell death, is down-regulated by vasoactive intestinal peptide in Th2 but not Th1 effectors

Following antigenic stimulation and differentiation, Th1 and Th2 effector cells contribute differently to cellular and humoral immunity. Vasoactive intestinal peptide (VIP) induces Th2 responses by promoting Th2 differentiation and survival. In this study, we investigate the mechanisms for the protective effect of VIP against activation-induced cell death (AICD) of Th2 effectors. Surprisingly, microarray and protein data indicate that VIP prevents the up-regulation of granzyme B (GrB) in Th2 but not Th1 effectors. This is the first report of GrB expression in Th cells and of its involvement in activation-induced apoptosis. The enhanced responsiveness of Th2 cells to VIP is probably due to the higher expression of VIP receptors. The effect of VIP on Th2 survival and GrB expression is mediated through the VIP receptors 1 and 2 and cAMP signaling through exchange protein activated by cAMP and, to a lesser degree, protein kinase A. In addition to effects on GrB, VIP also down-regulates Fas ligand (FasL) and perforin (Pfr) expression. The extrinsic Fas/FasL pathway and the intrinsic GrB-dependent pathway act independently in inducing AICD. The mechanisms by which GrB induces cell death in Th1/Th2 effectors include both fratricide and suicide. Fratricide killing, prevalent in wild-type cells, is calcium and Pfr dependent, whereas the cell death of Pfr-deficient Th cells involves Fas and GrB but is calcium independent. This study identifies GrB as a new significant player in Th1/Th2 AICD and characterizes two mechanisms for the protective effect of VIP on Th2 survival, i.e., the down-regulation of GrB and FasL expression.     

A study of quercetin effects on phospholipid membranes containing cholesterol using Laurdan fluorescence

Quercetin (QCT) is an important bioactive natural compound found in numerous edible plants. Since the lipid bilayer represents an essential compound of the cell membrane, QCT's direct interaction with this structure is of great interest. Therefore, we proposed to study the effects of QCT on DMPC liposomes containing cholesterol (Chol), and for this purpose Laurdan fluorescence was used. As a fluorescent probe, Laurdan is able to detect changes in membrane phase properties. When incorporated in lipid bilayers, Laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. The main tool for quantifying QCT's effects on phospholipid membranes containing Chol has been the analysis, the decomposition of Laurdan emission spectra in sums of two Gaussian functions on energy. This kind of approach has allowed good analysis of the balance between the two emitting states of Laurdan. Our results show that both Laurdan emission states are present to different extents in a wide temperature range for DMPC liposomes with Chol. QCT is decreasing the phase transition temperature in pure DMPC liposomes as proved by generalized polarization (GP) values. QCT also quenches Laurdan fluorescence, depending on the temperature and the presence of Chol in the membrane. Stern-Volmer constants were calculated for different lipid membrane compositions, and the conclusion was that the relaxed state favors the nonradiative transitions of the fluorophore.     

Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.     

A two-gate model for the ryanodine receptor with allosteric modulation by caffeine and quercetin

We have developed a model of the tetrameric ryanodine receptor--the calcium channel of the sarcoplasmic reticulum. The model accurately describes published experimental data on channel activity at various concentrations of Ca2+, caffeine and quercetin. The proposed mechanisms involve allosteric regulation of Ca2+ affinity by both caffeine and quercetin, and the existence of two independent, A- and I-gates controlled by Ca2+ binding to an activating and an inhibitory module of the receptor. There are four different configurations of the receptor that affect ligand binding to the activation module, but not to the inhibition module. Consequently, there are four kinetic modes for the A-gate and one mode for the I-gate. At a certain moment, the receptor can be in any of the four possible conformations with equal probability. By fitting the data we are able to derive ligand affinities and Hill coefficients, to describe the observation that quercetin is an activating agent stronger than caffeine, and that caffeine and quercetin activate the channel at very low Ca2+ concentration (approximately 10(-11) M). We predict that the activation regime at saturating caffeine or quercetin should present four distinct regions at increasing Ca2+, corresponding to the four different gating modes. Another interesting prediction is the enlargement of the activity domain toward higher Ca2+ concentrations in the presence of caffeine or quercetin.     

The role of water in the extracellular half channel of bacteriorhodopsin.

The changes in the photocycle of the wild type and several mutant bacteriorhodopsin (D96N, E204Q, and D212N) were studied on dried samples, at relative humidities of 100% and 50%. Samples were prepared from suspensions at pH approximately 5 and at pH approximately 9. Intermediate M with unprotonated Schiff base was observed at the lower humidity, even in the case where the photocycle in suspension did not contain this intermediate (mutant D212N, high pH). The photocycle of the dried sample stopped at intermediate M1 in the extracellular conformation; conformation change, switching the accessibility of the Schiff base to the cytoplasmic side, and proton transport did not occur. The photocycle decayed slowly by dissipating the absorbed energy of the photon, and the protein returned to its initial bacteriorhodopsin state, through several M1-like substates. These substates presumably reflect different paths of the proton back to the Schiff base, as a consequence of the bacteriorhodopsin adopting different conformations by stiffening on dehydration. All intermediates requiring conformational change were hindered in the dried form. The concentration of intermediate L, which appears after isomerization of the retinal from all-trans to 13-cis, during local relaxation of the protein, was unusually low in dried samples. The lack of intermediates N and O demonstrated that the M state did not undergo a change from the extracellular to the cytoplasmic conformation (M1 to M2 transition), as already indicated by Fourier transform infrared spectroscopy, quasielastic incoherent neutron scattering, and electric signal measurements described in the literature.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide stimulate the induction of Th2 responses by up-regulating B7.2 expression.

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two structurally related neuropeptides produced within the lymphoid microenvironment, modulate several immunologic functions. We have recently demonstrated that VIP and PACAP enhance the macrophage costimulatory activity for naive CD4+ T cells exposed to allogeneic or anti-CD3 stimuli through the differential regulation of the B7 costimulatory molecules. In this study, we report on the role of VIP and PACAP on macrophage B7 expression and costimulatory function for Ag-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo. VIP and PACAP up-regulate the costimulatory activity of macrophages for Ag-primed CD4+ T cells. VIP-/PACAP-treated macrophages gain the ability to induce Th2-type cytokines such as IL-4 and IL-5 and reduce Th1-type cytokines such as IFN-gamma and IL-2. In vivo administration of VIP or PACAP in Ag-immunized mice reduce the numbers of IFN-gamma-secreting cells and enhance the numbers of IL-4-secreting cells. One of the consequences of the VIP-/PACAP-induced shift in cytokine profile is a change in the Ag-specific Ig isotype, increasing IgG1 and decreasing IgG2a levels. Finally, the preferential differentiation into Th2 effector cells after Ag stimulation induced by VIP-/PACAP-treated macrophages is mediated through the up-regulation of B7.2 expression.     

Bacterial transporters: Charge translocation and mechanism

A comparative review of the electrophysiological characterization of selected secondary active transporters from Escherichia coli is presented. In melibiose permease MelB and the Na⁺/proline carrier PutP pre-steady-state charge displacements can be assigned to an electrogenic conformational transition associated with the substrate release process. In both transporters cytoplasmic release of the sugar or the amino acid as well as release of the coupling cation are associated with a charge displacement. This suggests a common transport mechanism for both transporters. In the NhaA Na⁺/H⁺ exchanger charge translocation due to its steady-state transport activity is observed. A new model is proposed for pH regulation of NhaA that is based on coupled Na⁺ and H⁺ equilibrium binding.