Limited contribution of Toll-like receptor 2 and 4 to the host response to a fungal infectious pathogen, Cryptococcus neoformans

The present study was designed to elucidate the role of Toll-like receptor (TLR) 2 and TLR4 in the host response to Cryptococcus neoformans. Both TLR2 knockout (KO) and TLR4KO mice produced interleukin-1beta (IL-1beta), IL-6, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha) in sera and cleared this fungal pathogen from infected lungs at a comparable level to control littermate (LM) mice. Synthesis of these cytokines was not significantly different in the lungs of these KO mice and LM mice, although IL-1beta, IL-6 and IL-12p40 tended to be lower in TLR2KO, but not TLR4KO, mice than in controls. In addition, there was no significant reduction detected in the synthesis of IL-12 and TNF-alpha by bone marrow-derived dendritic cells from TLR2KO and TLR4KO mice upon stimulation with live yeast cells. Finally, HEK293 cells expressing either TLR2/dectin-1 or TLR4/MD2/CD14 did not respond to C. neoformans in the activation of nuclear factor kappa B (NFkappaB) detected by a luciferase assay. Our results suggest that TLR2 and TLR4 do not or only marginally contribute to the host and cellular response to this pathogen.     

A protein kinase signal-responsive gene carrier modified RGD peptide

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using a cationic polymer. However, this polymer alone cannot deliver any gene into living cells. In the present work, we modified the signal-responsive polymer to the RGD peptide for the introduction of a polymer/DNA complex into living cells and succeeded in regulating the gene expression responding to intracellular PKA activation.     

P2X7 receptors regulate NKT cells in autoimmune hepatitis

Adenine nucleotides induce danger signals in T cells via purinergic receptors, raising the question whether they exert similar effects on innate immunity. Here we show that micromolar concentrations of nicotinamide adenine dinucleotide (NAD) induce a rapid increase of annexin V staining in NKT cells in vitro, a response that requires expression of P2X(7)Rs. Consistent with this result, treatment of mice with NAD causes a temporary decrease of NKT cells in the liver and protects from Con A- and alpha-galactosylceramide-induced hepatitis, both of which require functional NKT cells. Resistance to liver injury is associated with decreased cytokine production by NKT cells in NAD-treated mice. In contrast, when NAD is injected into Con A- or alpha-galactosylceramide-primed mice, liver injury is exacerbated and cytokine production by NKT cells is increased. This effect is caused by P2X(7)R-mediated stimulation of activated NKT cells. In agreement, mice lacking P2X(7)Rs on lymphocytes suffer reduced liver injury, and animals lacking ADP-ribosyltransferase, the enzyme that uses NAD to attach ADP-ribosyl groups to cell surfaces, are also resistant to Con A-induced hepatitis. These results prompt the conclusion that engagement of P2X(7)Rs on NKT cells inhibits naive, while stimulating activated cells, resulting in suppression or stimulation of autoimmune hepatitis.     

Involvement of Kupffer cells in lipopolysaccharide-induced rapid accumulation of platelets in the liver and the ensuing anaphylaxis-like shock in mice

Intravenous injection of Klebsiella O3 lipopolysaccharide (LPS) into BALB/c mice induces an anaphylaxis-like shock within minutes. Using 5-hydroxytryptamine as a marker for platelets, we previously suggested that a rapid platelet accumulation in the liver and lung precedes the shock, and that a complement-dependent platelet-degradation is involved in the shock. Here, we examined (i) the effect of platelet-depletion (using an anti-platelet monoclonal antibody) on the shock and (ii) the contribution of macrophages to the platelet-accumulation in those organs. LPS-induced platelet-accumulations in the liver and lung were confirmed by immunostaining. In platelet-depleted mice, the shock was largely prevented. The number of F4/80-positive macrophages was much greater in liver than in lung, and the hepatic macrophages were largely lost in mice given clodronate-encapsulated liposomes. In mice treated with such liposomes, both the LPS-induced accumulation of platelets in the liver (but not in the lung) and the shock were largely prevented, and repopulation of hepatic macrophages restored these LPS-induced responses. These results suggest that (i) platelets are indeed involved in the shock, (ii) Kupffer cells mediate the hepatic platelet accumulation, and (iii) preventing this hepatic accumulation can largely prevent rapid shock being induced by LPS (at the dose used here).     

Ellagic acid in pomegranate suppresses resistin secretion by a novel regulatory mechanism involving the degradation of intracellular resistin protein in adipocytes

Resistin, an adipocytokine, is considered the link between obesity and type 2 diabetes. Pomegranate is a rich source of compounds used to treat metabolic diseases including type 2 diabetes. In this study, we found that consumption of pomegranate fruit extract (PFE) predominantly reduced the serum resistin levels in ovariectomized mice, an animal model with elevated resistin levels in serum and upregulated resistin mRNA expression in white adipose tissue. Moreover, the PFE significantly reduced the secretion and intracellular protein levels of resistin in differentiated murine 3T3-L1 adipocytes, but it did not alter resistin mRNA expression. When de novo protein synthesis was inhibited by the protein synthesis inhibitor cycloheximide, the intracellular resistin protein levels were drastically reduced by the PFE, suggesting that the PFE promoted the degradation of resistin at the protein level. We also found that ellagic acid (EA), a main component of pomegranate, had the same effects on the secretion and intracellular protein level of resistin. These results suggest that EA in pomegranate suppresses resistin secretion by a novel mechanism involving the degradation of intracellular resistin protein in adipocytes.     

Drawbacks of the ancient RNA-based life-like system under primitive earth conditions

Following the discovery of ribozymes, the “RNA world” hypothesis has become the most accepted hypothesis concerning the origin of life and genetic information. However, this hypothesis has several drawbacks. Verification of the hypothesis from different viewpoints led us to proposals from the viewpoint of the hydrothermal origin of life, solubility of RNA and related biopolymers, and the possibility of creating an evolutionary system comparable to the in vitro selection technique for functional RNA molecules based on molecular biology.     

Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants

The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC-MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (Arabidopsis and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells.     

Sphingosine kinase 1 mediation of expression of the anaphylatoxin receptor C5L2 dampens the inflammatory response to endotoxin

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk1(-/-)) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk1(-/-) mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk1(-/-) mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury.