Adiponectin induces dendritic cell activation via PLCγ/JNK/NF-κB pathways, leading to Th1 and Th17 polarization

Adiponectin (APN) is a crucial regulator for many inflammatory processes, but its effect on Th cell-mediated responses has not been fully understood. Thus, we investigated the immune-modulatory effects of APN on dendritic cells (DCs) controlling Th cell polarization. APN induced maturation and activation of DCs, as demonstrated by the increased expression of MHC class II, costimulatory molecules in both mouse and human DCs, and it significantly enhanced production of proinflammatory cytokines. APN triggered degradation of IκB proteins, nuclear translocation of NF-κB p65 subunit, and phosphorylation of MAPKs in DCs. Pretreatment with a phospholipase C (PLC)γ inhibitor and a JNK inhibitor suppressed IL-12 production and NF-κB binding activity. Additionally, PLCγ inhibitor downregulated phosphorylation of JNK, indicating that PLCγ and JNK may be upstream molecules of NF-κB. Importantly, APN-treated DCs significantly induced both Th1 and Th17 responses in allogeneic CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb to the cocultures abolished the secretion of IFN-γ, whereas the blockage of IL-23 and IL-1β suppressed APN-induced IL-17 production. Immunization of mice with OVA-pulsed, APN-treated DCs efficiently led to Ag-specific Th1 and Th17 cell responses. Taken together, these results demonstrated that APN effectively induced activation of DCs through PLCγ/JNK/NF-κB-signaling pathways, leading to enhanced Th1 and Th17 responses.     

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions

The H2O2-catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site-directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D-hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)- and T90A-hPrxI, the T90D-hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT-hPrxI.     

Energy-dependent exchange of K+ in heart mitochondria. K+ influx.

The tuberculostatic and carcinogenic drug isonicotinic acid hydrazide (“isoniazid”) is oxidized to pyridine-4-carboxaldehyde by the horseradish peroxidase/Mn²⁺/O₂ system. Eosin and related sensitizers greatly accelerate the reaction and generate light detectable with the liquid scintillation counter or with the photon counter. If the isoniazid concentration is raised, the rate and extent of O₂ uptake are increased, but above a certain concentration of isoniazid, emission is reduced and even suppressed. The strong quencher of triplet eosin, potassium ferricyanide, abolished both effects of eosin, that is, catalysis and light emission. Superoxide dismutase at high concentrations partially suppressed the emission and almost totally removed the catalytic effect. It is tentatively proposed that the isoniazid/peroxidase/Mn²⁺/O₂ system efficiently produces the aldehyde in the triplet state, which in turn transfers energy to eosin. Because of the presence of oxygen, only a small yield of triplet eosin is obtained and only a small fraction of these triplet eosin molecules is able to react with isoniazid. Nevertheless, it will contribute efficiently to a cyclic process of oxidation of the isoniazid.     

The Universal Stress Protein UspC Scaffolds the KdpD/KdpE Signaling Cascade of Escherichia coli under Salt Stress

The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K⁺-transport system KdpFABC in response to K⁺ limitation or salt stress. Under K⁺ limiting conditions the Kdp system restores the intracellular K⁺ concentration, while in response to salt stress K⁺ is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K⁺, so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro reconstructed signaling cascade (KdpD→KdpE→DNA) resulting in phosphorylation of KdpE at a K⁺ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K⁺ limitation. Biochemical studies revealed that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE∼P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial two-component system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.     

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.     

Protoplast Fusion of Lactobacillus casei

Lactobacillus casei ATCC 7469 was successfully converted to protoplasts by treatment with endo-7V-acetyl muramidase in sucrose phosphate buffer. For full hydrolysis of cell walls, a high concentration of sucrose and a cold shock were necessary. Mg²⁺ ions enhanced the stability of protoplasting cells. The cell wall regeneration of protoplasts was more effective on gelatin-induced regeneration medium than with the soft overlay method. The optimal concentration of gelatin was 2.5%. The frequency of regeneration was found to be about 6% for the protoplast prepared by enzyme treatment for 20 min. The mutants having streptomycin resistance and rifampicin resistance, as selection markers for the detection of fusion, were isolated by UV irradiation and NTG treatment. These mutants were stable for at least several transfers. Protoplast fusion was carried out using PEG (50% solution of polyethyleneglycol, M.W. 6,000). The frequency of protoplast fusion was found to be about 10^-5.     

Identification of Staphylococcus xylosus isolated from C57BL/6J-Nos2(tm1Lau) mice with dermatitis

Coagulase negative staphylococci (CNS) are significant pathogens, particularly in medical device related infections and in immunocompromised patients. Five CNS strains were isolated from 5 NOS2 knockout mice with dermatitis. Histologically, granulomatous dermatitis was found in the skin around the ear with epidermal ulceration. Dermal lesions included pustules, necrosis, and accumulations of neutrophils and macrophages. Isolates of the bacterial strains were identified to be Staphylococcus xylosus by the API STAPH kit and 16S-23S intergenic transcribed spacer-PCR. These results demonstrate the potential of this organism to be an opportunistic pathogen in immunocompromised mice.