Protective Effect of Indole-3-Pyruvate against Ultraviolet B-Induced Damage to Cultured HaCaT Keratinocytes and the Skin of Hairless Mice

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm²) and maintained with or without test compounds (1–25 mM). In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (100 µmol) and exposed to UVB light (1 J/cm²) for two times. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.     

Adipose Hypothermia in Obesity and Its Association with Period Homolog 1, Insulin Sensitivity,and Inflammation in Fat

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H₂O₂, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.     

Induction of cytotoxicity by chlorogenic acid in human oral tumor cell lines.

Millimolar concentrations of chlorogenic acid (CGA) showed higher cytotoxic activity against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines, as compared with that against human gingival fibroblast (HGF). The cytotoxic activity of CGA was significantly reduced by catalase or CoCl2, but not affected by FeCl3 or CuCl2. ESR spectroscopy showed that higher (millimolar) concentrations of CGA produced radicals under alkaline conditions, acting as a prooxidant, whereas lower concentrations of CGA scavenged superoxide and hydroxyl radical. CGA produced large DNA fragments (as identified by slightly faster migrating band of DNA on agarose gel electrophoresis) and nuclear condensation (as demonstrated by Hoechst (No. 33258) staining) in tumor cell lines. Activation of caspase was demonstrated by staining with M30 monoclonal antibody, which reacts with degradation products of cytokeratin 18. Contact with CGA for at least 6 h was necessary for irreversible cytotoxicity induction. Pretreatment of the cells with caspase 3 inhibitor partially inhibited the cytotoxic action of CGA. These date suggest that CGA induces cytotoxicity in oral tumor cell lines, possibly by hydrogen peroxide-mediated oxidation mechanism.     

An endophyte of the tropical forage grass Brachiaria brizantha: isolating, identifying, and characterizing the fungus, and determining its antimycotic properties

Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C4 grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.     

Dendritic Cells from Spleen, Mesenteric Lymph Node and Peyer's Patch Can Induce the Production of Both IL-4 and IFN-γ from Primary Cultures of Naive CD4+ T Cells in a Dose-Dependent Manner

Dendritic cells (DCs) as antigen presenting cells can stimulate naive CD4⁺ T cells and initiate the primary immune response which controls Th1/Th2 development. It has been suggested that DCs derived from different tissues have distinct properties. We investigated whether DCs from mesenteric lymph nodes (MLN), Peyer's patches (PP) and spleen (SPL) could induce different responses of naive CD4⁺ T cells to varying doses of antigen by using a co-culture system of DCs and T cells. DCs from each tissue induced IL-4 secretion from naive CD4⁺T cells in the presence of low dose antigenic peptide, and induced IFN-γ production at high doses of antigen. When purified CD11c⁺/B220^? DCs were used, MLN-derived DCs induced a higher amount of IFN-γ secretion from naive CD4⁺ T cells, compared with SPL-derived DCs. We could not detect large differences in the expressions of costimulatory molecules on the surface of these two populations of DCs. On the other hand, we found that large amounts of IL-12 were secreted from MLN DCs in an antigen dose-dependent fashion. In conclusion, DCs from SPL, MLN and PP can induce the production of both IL-4 and IFN-γ from naive CD4⁺ T cells, depending on antigen dose. MLN-derived CD11c⁺/B220^? DCs induce higher IFN-γ production from naive CD4⁺ T cells than SPL-derived DCs, through efficient IL-12 secretion.     

Immunochemical detection of arachidonoyl-preferential phospholipase A2.

Monoclonal antibodies were raised against rabbit platelet cytosolic arachidonoyl-preferential phospholipase A2. The antibodies precipitated the arachidonoyl-preferential phospholipase A2 activity in the soluble fraction of a rabbit platelet lysate in combination with an immobilized anti-mouse immunoglobulin antibody, and reacted predominantly with a protein exhibiting a molecular weight of approximately 88,000 on immunoblotting analysis. All three antibodies established so far reacted with human platelet arachidonoyl-preferential phospholipase A2 as effectively as the rabbit platelet enzyme. One of them reacted with the rat platelet arachidonoyl-preferential enzyme, whereas none of them reacted with rabbit platelet secretory 14-kDa group II phospholipase A2. The existence of an immunologically related phospholipase A2 was further shown in rabbit granulocytes, brain, lung, and liver, rat and mouse mast cells, and human monocytoma U937 cells. Thus, an arachidonoyl-preferential phospholipase A2 with similar structural properties appeared to be expressed in a variety of cells and tissues.     

A Crucial Role for CDC42 in Senescence-Associated Inflammation and Atherosclerosis

Risk factors for atherosclerosis accelerate the senescence of vascular endothelial cells and promote atherogenesis by inducing vascular inflammation. A hallmark of endothelial senescence is the persistent up-regulation of pro-inflammatory genes. We identified CDC42 signaling as a mediator of chronic inflammation associated with endothelial senescence. Inhibition of CDC42 or NF-κB signaling attenuated the sustained up-regulation of pro-inflammatory genes in senescent human endothelial cells. Endothelium-specific activation of the p53/p21 pathway, a key mediator of senescence, also resulted in up-regulation of pro-inflammatory molecules in mice, which was reversed by Cdc42 deletion in endothelial cells. Likewise, endothelial-specific deletion of Cdc42 significantly attenuated chronic inflammation and plaque formation in atherosclerotic mice. While inhibition of NF-κB suppressed the pro-inflammatory responses in acute inflammation, the influence of Cdc42 deletion was less marked. Knockdown of cdc-42 significantly down-regulated pro-inflammatory gene expression and restored the shortened lifespan to normal in mutant worms with enhanced inflammation. These findings indicate that the CDC42 pathway is critically involved in senescence-associated inflammation and could be a therapeutic target for chronic inflammation in patients with age-related diseases without compromising host defenses.     

Thermostabilization of protein A-luciferase fusion protein by single amino acid mutation

A gene expression plasmid, pMALU7, for coding a fusion protein between protein A (SpA) and mutated firefly luciferase (Luc), was constructed. The fused gene was expressed in Escherichia coli and the resulting protein (SpA-LucTS) was purified with affinity chromatography. By changing a single amino acid, from Glu to Lys at the position 354 in the luciferase moiety, the thermostability of luciferase was improved.