BBA - General Subjects (v.1820, #4)

Ginsenoside Rg1 promotes nonamyloidgenic cleavage of APP via estrogen receptor signaling to MAPK/ERK and PI3K/Akt by Chun Shi; Dong-dan Zheng; Li Fang; Fengming Wu; Wing Hang Kwong; Jie Xu (453-460).
The pathogenic accumulation of amyloid β peptide (Aβ), a natural occurring peptide processed from beta-amyloid precursor protein (APP), is considered to play a key role in the development of Alzheimer's disease (AD). Ginsenoside Rg1, an active component in ginseng, has been identified as a phytoestrogen and also found to be neuroprotective. However, it is unknown whether Rg1-induced estrogenic activity intervenes in APP processing, and improves memory performance.Using HT22 cells and SH-SY5Y cells stably expressing the Swedish mutant APP (APPsw), this study investigated whether Rg1 intervened in APP metabolism through estrogenic activity. Using the ovariectomized (OVX) rats to mimic age-related changes in postmenopausal females, this study also tested the long-term effect of Rg1 on APP metabolism.The in vitro study demonstrated that Rg1 increased extracellular secretion of soluble amyloid precursor protein α (sAPPα), enhanced α-secretase activity and decreased extracellular release of Aβ. These effects of Rg1 could be prevented by inhibitors of protein kinase C (PKC), Extracellular-Signal Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) and Phosphoinositide-3 kinase (PI3K)/Akt pathways. Inhibition of endogenous estrogen receptor (ER) activity abrogated Rg1-triggered release of sAPPα, increase of α-secretase activity, and activation of ERK and Akt signaling. In addition, Rg1 promoted phosphorylation of ERα at Ser118 residue. The in vivo study demonstrated that 8-week Rg1 treatment of OVX rats increased sAPPα levels and decreased Aβ content in the hippocampi, and improved the spatial learning and memory.Rg1 might be used to slow or prevent AD, in particular in postmenopausal females.► The estrogenic effect of ginsenoside Rg1 on APP metabolism. ► Rg1 promotes α-secretase cleavage of APP via ER signaling to MAPK/ERK and PI3K/Akt. ► Rg1 activated ER signaling via phosphorylation of ER at Ser118 residue.
Keywords: Ginsenoside Rg1; Amyloid precursor protein; Estrogen receptor; Extracellular-Signal Regulated Kinase/Mitogen-Activated Protein Kinase;

Nobiletin suppresses adipocyte differentiation of 3T3-L1 cells by an insulin and IBMX mixture induction by Kota Kanda; Kosuke Nishi; Ayumu Kadota; Sogo Nishimoto; Ming-Cheh Liu; Takuya Sugahara (461-468).
Nobiletin is a citrus flavonoid which possesses the flavone structure with six methoxy groups. Although nobiletin has been reported to display anti-inflammatory, anti-tumor, and anti-diabetes activities, its effect on adipocyte differentiation remained unclear. In the present study, we investigated the effect of nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes.3T3-L1 preadipocytes were treated with nobiletin under various differentiation conditions. The effect of nobiletin on adipocyte differentiation was evaluated by oil red O staining, real-time RT-PCR, and Western blotting.Nobiletin significantly suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes, upon induction with insulin together with a cAMP elevator such as 3-isobutyl-1-methylxanthine (IBMX), by downregulating the expression of the gene encoding peroxisome proliferator-activated receptor (PPAR) γ2. In addition, nobiletin decreased the phosphorylation of cAMP-response element-binding protein (CREB) and strongly enhanced the phophorylation of signal transducer and activator of transcription (STAT) 5.Nobiletin has a suppressive effect on the differentiation of preadipocytes into adipocytes when cells were induced with a general differentiation cocktail such as insulin, IBMX, and dexamethasone.► Nobiletin is a citrus flavonoid. ► Nobiletin suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes. ► Gene expression of PPARγ2 gene was down-regulated by nobiletin. ► STAT5 was strongly phosphorylated by nobiletin. ► Excessive activation of STAT5 appeared to suppress the PPARγ2 gene expression.
Keywords: Nobiletin; PPARγ; STAT5; 3T3-L1; cAMP; CREB;

Wharton's Jelly cells (WJCs) can be differentiated into adipocytes by cytoskeletal reorganisation in association with changes in the mechanical properties of cells.WJCs subjected to adipocyte induction were observed changes in the cell morphology and alterations in actin filament formation. Transfection with either small interfering RNAs (siRNAs) against formin-2 (FMN-2), tropomyosin-1 (Tm-1), caldesmon (CaD), and profilin (Pro) or a pcDNA6-gelsolin (GSN)-constructed vector in WJCs was used to establish their regulatory roles in controlling adipogenesis. Phenotypic transformation of the cell shape and changes in cell surface adhesion force were determined in WJCs after transformation.The levels of protein and mRNA expression of β-actin and several key actin binding ptoteins (ABPs) were decreased during the early stage of adipogenic induction but were recovered in the later induction. The siFMN-2, siTm-1, siCaD, and siPro gene knockdown in WJCs caused a widening of the cell shape, while WJCs overexpressing GSN retained a fibroblast cell shape. For both transformations, atomic force microscopy revealed alterations in the biomechanical signals on the cell surface. However, the adipogenic potency was increased after siFMN-2, siTm-1, siCaD, and siPro gene knockdown and decreased during GSN overexpression.siRNA gene knockdown of siFMN-2, siTm-1, siCaD, and siPro enhances the potency for WJCs commitment to adipocyte, while GSN overexpression modulates the PPAR-γ-independent pathway for the adipogenesis of WJCs.The phenotypic changes associated with decreased ABP gene expression are critical for regulating the adipogenic differentiation of WJCs through the temporal control of actin filament organisation.► Cytoskeletal remodelling is involved in governing the adipogenic induction of WJCs. ► Early downregulation of actin and ABPs is essential for the adipogenesis of WJCs. ► siRNA gene knockdown of CaD enhances the potency for WJCs commitment to adipocyte. ► GSN op regulates the PPAR-γ-independent pathway for the adipogenesis of WJCs.
Keywords: WJCs; ABPs; Small interfering RNAs; Overexpression; Atomic force microscopy;

Primary-amine oxidase (PrAO) catalyzes the oxidative deamination of endogenous and exogenous primary amines and also functions, in some tissues, as an inflammation-inducible endothelial factor, known as vascular adhesion protein-1. VAP-1 mediates the slow rolling and adhesion of lymphocytes to endothelial cells in a number of inflammatory conditions, including inflammation of the synovium.Glucosamine binding to the enzyme was assessed spectrofluorometrically and the kinetics of inhibition of PrAO were determined spectrophotometrically through the use of direct or coupled assays, in the presence of different substrates.Glucosamine is not a substrate for PrAO, but acts as a time-dependent inhibitor of PrAO activity, displaying mixed inhibition kinetics. The observed inhibition and binding were augmented in the presence of H2O2.Significant in vitro effects on PrAO require glucosamine in the millimolar concentration range and it is not clear at this stage whether a low but persistent level of PrAO inhibition might contribute to the anti-arthritic response.This work was aimed at characterizing the interactions of PrAO/VAP-1 with glucosamine, a widely used “over-the-counter” supplement for the treatment of osteoarthritis.Display Omitted► Primary-amine oxidase mediates the adhesion of lymphocytes during inflammation. ► Glucosamine is widely used as a supplement for the treatment of arthritis. ► We show glucosamine to be a time-dependent inhibitor of PrAO activity. ► In vitro inhibition and binding were augmented in the presence of H2O2. ► Limitation: millimolar glucosamine concentrations required for inhibition in vitro.
Keywords: Vascular adhesion protein-1 (VAP-1); Enzyme inhibition; Glucosamine; Arthritis;

d-ribose in glycation and protein aggregation by Yan Wei; Chan Shuai Han; Jun Zhou; Ying Liu; Lan Chen; Rong Qiao He (488-494).
d-ribose is a naturally occurring pentose monosaccharide present in all living cells and their microenvironments and is a key component of numerous biomolecules involved in many important metabolic pathways. It also participates in the glycation of proteins producing advanced glycation end products (AGEs) that lead to cell dysfunction and death. As recent studies show, ribosylation, a rapid process, causes protein aggregation in vitro and in vivo.This review summarizes the relationship between ribosylation, protein aggregation, cell death and cognitive impairments. d-ribose is active in glycation and induces protein aggregation, rapidly producing AGEs in vitro and in vivo.Ribosylation, leading to the production of significant amounts of AGEs both extracellularly and intracellularly, may be involved in cell dysfunction and subsequent cognitive impairments. This review may be a useful reference for studies on the pharmacokinetics of d-ribose action.► d-ribose is most active in glycation and induces protein aggregation. ► Ribosylation leads to AGEs production extracellularly and intracellularly. ► Taking d-ribose increases glycated blood protein though hypoglycemia is reported. ► Risks of long term administration of d-ribose should be taken into consideration.
Keywords: d-ribose; Glycation; Protein aggregation; Advanced glycation end product; Cognitive impairment;

Overexpression of SREBP-1 causes a repression of hepatic genes involved in phase II metabolism. In LDL receptor deficient (LDLR−/−) mice, active levels of SREBP-1 in the liver are increased. We investigated the hypothesis that LDLR−/− mice have increased concentrations of thyroid hormones in plasma due to a reduced hepatic glucuronidation.Female LDLR−/− and wild-type mice were used to study the effect of the LDLR−/− genotype on thyroid hormone metabolism.LDLR−/− mice had a higher concentration of nuclear SREBP-1, higher concentrations of thyroxine and triiodothyronine in plasma, a lower expression of relevant UGT1A isoforms, reduced activities of pNP-UGT, T3-UGT and T4-UGT and a lower mRNA and protein concentration of AhR in the liver than wild-type mice (P  < 0.05). Plasma concentration of TSH, mRNA concentrations of various genes involved in thyroid hormone synthesis in the thyroid, activity of deiodinase and mRNA concentrations of two thyroid hormone responsive genes, CYP7A1 and Na+/K+-ATPase, in the liver did not differ between both genotypes.This study shows that LDLR−/− mice have increased concentrations of thyroid hormones in plasma. This effect is probably due to an inhibition of thyroid hormone glucuronidation, which might be caused by down-regulation of UGT genes due to a reduced expression of AhR. However, with respect to plasma TSH concentration and expression of thyroid hormone responsive genes no overt hyperthyroidism was detected.LDL receptor deficiency leads to a reduced glucuronidation of thyroid hormones in the liver which causes a moderate increase of plasma thyroid hormone concentrations.► LDLR−/− mice have a reduced expression of AhR and UGTs in the liver. ► LDLR−/− mice have a diminished glucuronidation of thyroid hormones. ► LDLR−/− mice have increased plasma thyroid hormone concentrations. ► An interaction between SREBP-1, AhR and thyroid hormone metabolism is proposed.
Keywords: LDLR−/−; Thyroid hormone; SREBP-1; Aryl hydrocarbon receptor; UDP glucuronosyltransferase;

Minocycline exhibits anti-inflammatory properties independent of its antibiotic activity, ameliorating inflammatory responses in monocytes and macrophages. However, the mechanisms of minocycline anti-inflammatory effects are only partially understood.Human circulating monocytes were cultured in the presence of lipopolysaccharide (LPS), 50 ng/ml, and minocycline (10–40 μM). Gene expression was determined by RT-PCR, cytokine and prostaglandin E2 (PGE2) release by ELISA, protein expression, phosphorylation and nuclear translocation by Western blotting.Minocycline significantly reduced the inflammatory response in LPS-challenged monocytes, decreasing LPS-induced transcription of pro-inflammatory tumor-necrosis factor alpha (TNF-α), interleukin-1 beta, interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), and the LPS-stimulated TNF-α, IL-6 and PGE2 release. Minocycline inhibited LPS-induced activation of the lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), NF-κB, LPS-induced TNF-α factor (LITAF) and the Nur77 nuclear receptor. Mechanisms involved in the anti-inflammatory effects of minocycline include a reduction of LPS-stimulated p38 mitogen-activated protein kinase (p38 MAPK) activation and stimulation of the phosphoinositide 3-kinase (PI3K)/Akt pathway.We provide novel evidence demonstrating that the anti-inflammatory effects of minocycline in human monocytes include, in addition to decreased NF-κB activation, abrogation of the LPS-stimulated LOX-1, LITAF, Nur77 pathways, p38 MAPK inhibition and PI3K/Akt activation. Our results reveal that minocycline inhibits points of convergence of distinct and interacting signaling pathways mediating multiple inflammatory signals which may influence monocyte activation, traffic and recruitment into the brain.Our results in primary human monocytes contribute to explain the profound anti-inflammatory and protective effects of minocycline in cardiovascular and neurological diseases and may have direct translational relevance.► We study minocycline effects on LPS inflammation in primary human monocytes. ► We examine LPS-induction of cytokines, NF-κB, p38 MAPK, Akt, LOX-1, LITAF and Nur77. ► Minocycline significantly inhibits all LPS effects. ► Minocycline effects are partly the consequence of Akt activation. ► The results clarify novel mechanisms of minocycline anti-inflammatory effects.
Keywords: Minocycline; Lipopolysaccharide; Nuclear receptor; LITAF; Inflammation; Human monocytes;

Morphological changes and oxidative damage in Rett Syndrome erythrocytes by Lucia Ciccoli; Claudio De Felice; Eugenio Paccagnini; Silvia Leoncini; Alessandra Pecorelli; Cinzia Signorini; Giuseppe Belmonte; Giuseppe Valacchi; Marcello Rossi; Joussef Hayek (511-520).
Hypoxemia and increased oxidative stress (OS) have been reported in Rett Syndrome (RTT), a genetical neurodevelopmental disorder. Although OS and hypoxemia can lead to red blood cells (RBCs) shape abnormalities, no information on RBCs morphology in RTT exists. Here, RBCs shape was evaluated in RTT patients and healthy subjects as a function of OS markers, blood oxygenation, pulmonary gas exchange, and cardio-respiratory parameters.RBCs morphology was evaluated by Scanning Electron Microscopy. Intraerythrocyte and plasma non protein-bound iron (NPBI), esterified F2-Isoprostanes (F2-IsoPs), 4-HNE protein adducts (4-HNE PAs) were measured. Pulmonary oxygen gradients and PaO2 were evaluated by gas analyzers and cardiopulmonary variables by pulse oximetry. In RTT patients these parameters were assessed before and after ω-3 polyunsaturated fatty acids (ω-3 PUFAs) administration.Altered RBCs shapes (leptocytes) and increased NPBI were present in RTT, together with increased erythrocyte membrane esterified F2-IsoPs and 4-HNE PAs. Abnormal erythrocyte shapes were related to OS markers levels, pulmonary gas exchange, PaO2 and cardio-respiratory variables. After ω-3 PUFAs, a decrease of leptocytes was accompanied by a progressive increase in reversible forms of RBCs. This partial RBCs morphology rescue was related to decreased OS damage markers, improved pulmonary oxygen exchange, and cardiopulmonary physiology.These findings indicate that in RTT 1) RBCs shape is altered; 2) the OS-hypoxia diad is critical in generating altered RBCs shape and membrane damage; 3) ω-3 PUFAs are able to partially rescue RBCs morphology and the OS-derived damage.RBCs morphology is an important biosensor for OS imbalance and chronic hypoxemia.► RBCs shape is critical to RBCs function and rheological properties of blood. ► Chronic hypoxemia and oxidative stress are key factors in modulating RBCs shape. ► We show altered RBCs shape in a genetical neurologic disease, Rett syndrome. ► Abnormal RBCs shape is related to evidence of oxidative membrane damage and hypoxia. ► ω-3 PUFAs partially rescue RBCs shape, oxidative damage and hypoxia.
Keywords: Rett syndrome; Erythrocytes; Oxidative stress; Hypoxemia; F2-Isoprostanes; Non protein-bound iron;

Silencing of FAD synthase gene in Caenorhabditis elegans upsets protein homeostasis and impacts on complex behavioral patterns by Vania C. Liuzzi; Teresa A. Giancaspero; Elisabetta Gianazza; Cristina Banfi; Maria Barile; Carla De Giorgi (521-531).
FAD synthase is a ubiquitous enzyme that catalyses the last step of FAD biosynthesis, allowing for the biogenesis of several flavoproteins. In humans different isoforms are generated by alternative splicing, isoform 1 being localized in mitochondria. Homology searching in Caenorabditis elegans leads to the identification of two human FAD synthase homologues, coded by the single copy gene R53.1.The C. elegans R53.1 gene was silenced by feeding. The expression level of transcripts was established by semi-quantitative RT-PCR. Overall protein composition was evaluated by two-dimensional electrophoresis. Enzymatic activities were measured by spectrophotometry and oxygen consumption by polarography on isolated mitochondria.From R53.1 two transcripts are generated by trans-splicing. Reducing by 50% the transcription efficiency of R53.1 by RNAi results in a 50% reduction in total flavin with decrease in ATP content and increase in ROS level. Significant phenotypical changes are noticed in knock-down nematodes. Among them, a significant impairment in locomotion behaviour possibly due to altered cholinergic transmission. At biochemical level, impairment of flavoenzyme activities and of some KCN-insensitive oxygen-consuming enzymes is detected. At proteomic level, at least 15 abundant proteins are affected by R53.1 gene silencing, among which superoxide dismutases.For the first time we addressed the existence of different isoforms of FAD-metabolizing enzymes in nematodes. A correlation between FAD synthase silencing and flavoenzyme derangement, energy shortage and redox balance impairment is apparent. In this aspect R53.1-interfered nematodes could provide an animal model system for studying human pathologies with alteration in flavin homeostasis/flavoenzyme biogenesis. ► R53.1 gene is transcribed via a trans-splicing mechanism in two mRNAs in C.elegans. ► Significant phenotypical changes are observed upon gene silencing. ► Dysregulation of many enzymes is observed with the decreased content of FAD. ► R53.1-interfered nematodes could provide an animal model system for studying human pathologies involving FAD deficiency.
Keywords: FAD synthase; Trans-splicing; R53.1 gene; Caenorhabditis elegans; Mitochondria; Flavin cofactors;

Cells avoid major mitochondrial damage and energy failure during systemic inflammatory states, such as severe acute infections, by specific targeting of the inflammatory response and by inducing anti-inflammatory and anti-oxidant defenses. Recent evidence indicates that these cell defenses also include mitochondrial biogenesis and the clearance of damaged mitochondria through autophagy.This review addresses a group of transcriptional signaling mechanisms that engage mitochondrial biogenesis, including energy-sensing and redox-regulated transcription factors and co-activators, after major inflammatory events.Stimulation of the innate immune system by activation of toll-like receptors (TLR) generates pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α)and interleukin-1β (IL-1β), necessary for optimal host defense, but which also contribute to mitochondrial damage through oxidative stress and other mechanisms. To protect its energy supply, host cells sense mitochondrial damage and initiate mitochondrial biogenesis under the control of an inducible transcriptional program that also activates anti-oxidant and anti-inflammatory gene expression. This multifunctional network not only increases cellular resistance to metabolic failure, oxidative stress, and cell death, but promotes immune tolerance as shown in the graphical abstract.The post-inflammatory induction of mitochondrial biogenesis supports metabolic function and cell viability while helping to control inflammation. In clinical settings, patients recovering from severe systemic infections may develop transient immune suppression, placing them at risk for recurrent infection, but there may be therapeutic opportunities to enhance mitochondrial quality control that would improve the resolution of life-threatening host responses to such infections.Display Omitted► The innate immune response triggers a cycle of oxidative mitochondrial damage. ► The host cell activates the transcriptional program for mitochondrial biogenesis. ► The program concurrently activates anti-oxidant and anti-inflammatory defense genes. ► This network limits metabolic failure and apoptosis and promotes immune tolerance.
Keywords: Infection; Inflammasome; Innate immunity; Cell metabolism; Oxidative stress;

Characterization of a novel tyrosinase inhibitor, (2RS,4R)-2-(2,4-dihydroxyphenyl)thiazolidine-4-carboxylic acid (MHY384) by Yu Kyeong Han; Yun Jung Park; Young Mi Ha; Daeui Park; Ji Yeon Lee; Naree Lee; Jeong Hyun Yoon; Hyung Ryong Moon; Hae Young Chung (542-549).
We synthesized (2RS,4R)-2-(2,4-dihydroxyphenyl)thiazolidine-4-carboxylic acid (MHY384) as a potential tyrosinase inhibitor and investigated its antityrosinase activity.The structure of MHY384 was established using 1H and 13C NMR spectroscopy and mass spectral analyses. To investigate dual mechanisms of action of MHY384 for the inhibition of melanin synthesis, we confirmed the inhibitory effect of tyrosinase catalytic activity of MHY384. Then, we confirmed the inhibitory effect of MHY384 on transcription of tyrosinase mRNA through alpha-MSH-induced cAMP–PKA–MITF signaling. In addition, we supported the inhibitory mechanism of MHY384 against tyrosinase using a kinetic study and docking programs.To determine how MHY384 regulates melanogenesis, we measured melanin levels and expression of the genes for microphthalmia-associated transcription factor (MITF) and tyrosinase in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 melanoma cells. MHY384 potently inhibited tyrosinase activity and melanin production in B16F10 melanoma cells. Through docking models, we were able to construct the tertiary structure of mushroom tyrosinase and simulate its docking with MHY384. The result supports that MHY384 strongly interacts with tyrosinase residues in the active site and it can directly inhibit tyrosinase. To investigate additional mechanisms of action of MHY384, we confirmed that the inhibition of tyrosinase activity was found to be due to the modulation of the expression of tyrosinase and its transcription factor, MITF, through cAMP, which regulates protein kinase A.This study strongly indicates that the depigmenting effect of MHY384 results from the down-regulation of MITF and tyrosinase through direct tyrosinase inhibition.Our findings suggest that MHY384 can be an effective skin-whitening agent.Display Omitted► We describe the design, synthesis and biological activities of MHY384. ► The depigmentary effect of MHY384 results from down-regulation of MITF. ► MHY384 can suppress the production of melanin. ► MHY384 can be an effective skin-whitening agent.
Keywords: Antityrosinase activity; Melanogenesis; Microphthalmia-associated transcription factor; Cyclic adenosine monophosphate; Skin-whitening agent; Docking analysis;