Effects of 15-deoxy-∆<Superscript>12,14</Superscript>-prostaglandin J<Subscript>2</Subscript> on the production of IL-8 and the expression of Toll-like receptor 2 in human primary keratinocytes stimulated with lipopolysaccharide

15-deoxy-∆^12,14-prostaglandin J₂ (15d-PGJ₂) is an anti-inflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We investigated the effects of 15d-PGJ₂ on the production of IL-8 and on the expression of Toll-like receptors (TLRs) 2 in human primary keratinocytes stimulated with lipopolysaccharide (LPS). Cell proliferation was analyzed using the MTT assay, TLR2 and -4 mRNA expression was detected by RT–PCR, and IL-8 production and NF-κB p65 activities were determined by ELISA. LPS and 15d-PGJ₂ did not influence the proliferation rate at low concentrations (0.5 and 2.0 μM) in keratinocytes, and showed toxicity at high concentrations (5.0 μM). LPS, compared with control, induced the expression of TLR2 mRNA, increased IL-8 production, and enhanced NF-κB activity. 15d-PGJ₂ decreased TLR2 mRNA, increased IL-8 production, and suppressed NF-κB activity. Costimulation with LPS and 15d-PGJ₂, compared with LPS stimulation alone, decreased TLR2 mRNA (1.8-fold), increased IL-8 production (1.8-fold at 0.5 μM and 3.7-fold at 2.0 μM), and inhibited NF-κB activity (3.3-fold at 0.5 μM and 5.1-fold at 2.0 μM). TLR4 mRNA was not expressed in primary keratinocytes. These results suggest that 15d-PGJ₂ suppresses TLR2 expression and that it up-regulates the production of IL-8 by inhibiting the NF-κB signaling pathway in primary keratinocytes. Thus, 15d-PGJ₂ can have both anti- and pro-inflammatory effects, and 15d-PGJ₂-mediated IL-8 up-regulation is related to the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways.     

Effects of Adrenalectomy and Replacement Therapy of Corticosterone on Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

Newly generated neurons in the dentate gyrus differentiate into mature granule cells. In the present study, we observed the effects of adrenalectomy (ADX) and corticosterone replacement therapy (CRT) on cell death, cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG). For this, the animals received vehicle or CRT after ADX, and were sacrificed 5 or 42 days later. Plasma corticosterone levels were very low in the adrenalectomized groups, whereas CRT after ADX significant increased serum corticosterone levels at 42 days, not 5 days, after ADX. ADX induced some neuronal damage in the dentate gyrus at 5 days post-ADX. CRT did not significantly reduce the neuronal damage at 5 days post-ADX; however, neuronal damage was not shown at 42 post-ADX with CRT. Ki67 (a marker for cell proliferation) and doublecortin (DCX, a marker for neuronal differentiation) immunoreaction was detected in the SZDG. ADX transiently increased cell proliferation and neuroblast differentiation 5 days after ADX, not 42 days, after ADX, and the CRT 42 days after ADX prominently decreased cell proliferation and neuroblast differentiation in the dentate gyrus. These results suggest that adrenal corticosteroid hormone is not essential for cell proliferation and neuroblast differentiation in long-term period after ADX.     

Comparison of Glucocorticoid Receptor and Ionized Calcium-Binding Adapter Molecule 1 Immunoreactivity in the Adult and Aged Gerbil Hippocampus Following Repeated Restraint Stress

Stress leads to changes in homeostasis and internal balance of the body and is known to be one of important factors in the development of several diseases. In the present study, we investigated changes in glucocorticoid receptor (GR) and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity in the adult and aged gerbil hippocampus after chronic restraint stress. Serum corticosterone level was much higher in both the stress-groups than the control groups. No neuronal death was found in all hippocampal subregions of the adult and aged gerbil after chronic restraint stress. GR immunoreactivity was decreased in both the adult and aged groups after repeated restraint stress; however, GR immunoreactivity in the adult-stress-group was decreased much more than that in the aged-stress-group. Iba-1 immunoreactive microglia were hypertrophied and activated in the adult group after repeated restraint stress; in the aged-stress-group, there was no any significant change in Iba-1 immunoreactive microglia. In brief, level of GR, not Iba-1, in the hippocampus was much decreased in the adult gerbil compared to the aged gerbil following chronic restraint stress.     

Vascular-specific activity of the Arabidopsis carotenoid cleavage dioxygenase 7 gene promoter

Carotenoid cleavage dioxygenases (CCDs) are involved in the production of diverse apocarotenoids including phytohormones, the visual molecules and the aromatic volatile compounds derived from carotenoids. Here, we examined the spatial expression of four of the CCD genes (AtCcd1, 4, 7 and 8) among the nine members of this family in Arabidopsis by RT-PCR. We found that the AtCcd7 gene showed strong expression in seeds. However, the promoter activity of the 1,867-bp 5′-upstream region of this gene exhibited a vascular specificity at all developmental stages throughout the transgenic Arabidopsis plants tested. The strength of the AtCcd7 promoter was also found to be lower than that of the 35S promoter by about 60%. The whole body expression of the β-glucuronidase (GUS) reporter gene driven by the AtCcd7 promoter in Arabidopsis plants was confirmed in different organs by RT-PCR and GUS enzymatic assays. Histochemical GUS staining further revealed that the AtCcd7 promoter has utility in limiting the expression of target genes to the vascular tissues in all plant organs such as the leaf, stem, root, flower and seed.     

Crystallization and preliminary X-ray crystallographic analysis of CTX-M-15, an extended-spectrum β-lactamase conferring worldwide emerging antibiotic resistance

CTX-M-15, an extended-spectrum β-lactamase emerging worldwide, hydrolyzes lactam ring of β-lactam antibiotics, and thus causes therapeutic failure and a lack of eradication of pathogenic bacteria by third-generation β-lactams. Therefore, the enzyme is a potential target for developing agents against pathogens isolated from patients suffering from nosocomial infections. The CTX-M-15 protein was purified and crystallized at 298 K. X-ray diffraction data from CTX-M-15 crystal have been collected to 1.46 ? resolution using synchrotron radiation. The crystal of CTX-M-15 belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.50, b = 44.23, and c = 116.92 ?. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 41.26%.     

Estrogenic activities of Psoralea corylifolia L. seed extracts and main constituents

Estrogenic activities of ethanol extract and its active components from Psoralea corylifolia L. were studied using various in vitro assays. The main components from ethanol extract were analyzed to be bakuchiol, psoralen, isobavachalcone, isobavachromene, and bavachinin. In a fractionation procedure, hexane and chloroform fractions showed estrogenic activity in yeast transactivation assay and E-screen assay. In yeast transactivation assay, ethanol extract, hexane, and chloroform fractions showed significantly higher activities at a concentration of 1.0 ng/ml, and bakuchiol at the concentration of 10⁻⁶ M was showed the highest activity, especially, which was higher than genistein at the same concentration. In E-screen assay, cell proliferation of bakuchiol (10⁻⁶ M) showed similar estrogenic activity with genistein (10⁻⁶ M). In ER binding assay, bakuchiol displayed the strongest ER-binding affinity (IC₅₀ for ERα = 1.01 × 10⁻⁶ M, IC₅₀ for ERβ = 1.20 × 10⁻⁶ M) and bakuchiol showed five times higher affinity for ERα than for ERβ.     

Novel enzymatic production of trehalose from sucrose using amylosucrase and maltooligosyltrehalose synthase-trehalohydrolase

Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is an important non-reducing disaccharide used in the food industry due to its mild sweetness (45% that of sucrose), low cariogenicity, high glass transition temperature, low hygroscopicity, and protein protection properties. In this study, we accomplished the production of trehalose from sucrose as a sole substrate using a novel dual-enzyme system, in which amylosucrase (ASase) and maltooligosyltrehalose synthase-trehalohydrolase (MTSH) fusion enzyme were employed. The biotransformation of sucrose to trehalose was confirmed by high-performance anion-exchange chromatography (HPAEC) analysis. Trehalose was successfully produced by both simultaneous and sequential methods by using ASase and MTSH. A higher trehalose production yield (3.15 ± 0.83 mM trehalose/20 mM sucrose) was observed in the sequential method than the simultaneous method (1.43 ± 0.14 mM trehalose/20 mM sucrose), indicating that the production of maltooligosaccharides from sucrose by ASase was an important step in the biosynthesis of trehalose.     

Isolation and characterization of IgG1 with asymmetrical Fc glycosylation

N-glycosylation of immunoglobulin G (IgG) at asparigine residue 297 plays a critical role in antibody stability and immune cell-mediated Fc effector function. Current understanding pertaining to Fc glycosylation is based on studies with IgGs that are either fully glycosylated [both heavy chain (HC) glycosylated] or aglycosylated (neither HC glycosylated). No study has been reported on the properties of hemi-glycosylated IgGs, antibodies with asymmetrical glycosylation in the Fc region such that one HC is glycosylated and the other is aglycosylated. We report here for the first time a detailed study of how hemi-glycosylation affects the stability and functional activities of an IgG1 antibody, mAb-X, in comparison to its fully glycosylated counterpart. Our results show that hemi-glycosylation does not impact Fab-mediated antigen binding, nor does it impact neonatal Fc receptor binding. Hemi-glycosylated mAb-X has slightly decreased thermal stability in the CH2 domain and a moderate decrease (～20%) in C1q binding. More importantly, the hemi-glycosylated form shows significantly decreased binding affinities toward all Fc gamma receptors (FcγRs) including the high-affinity FcγRI, and the low-affinity FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB. The decreased binding affinities to FcγRs result in a 3.5-fold decrease in antibody-dependent cell cytotoxicity (ADCC). As ADCC often plays an important role in therapeutic antibody efficacy, glycosylation status will not only affect the antibody quality but also may impact the biological function of the product.     

Neuroprotective effects of overexpressed cyclophilin B against Aβ-induced neurotoxicity in PC12 cells

Accumulated amyloid-β (Aβ) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aβ toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aβ_25-35 mediates apoptotic cell death. Aβ_25-35-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aβ_25-35 toxicity. We showed that Aβ_25-35-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aβ_25-35-induced toxicity. Consequently, these findings suggest that CypB may protect against Aβ toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway.