Check out our New Publishers' Select for Free Articles

BBA - General Subjects (v.1820, #1)

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-viii).

Effects of resveratrol on HepG2 cells as revealed by¹H-NMR based metabolic profiling by Mara Massimi; Alberta Tomassini; Fabio Sciubba; Anatoli P. Sobolev; Laura Conti Devirgiliis; Alfredo Miccheli (pp. 1-8).

Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis.We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a¹H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways.Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation.Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation.NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.► NMR-based metabolomics of culture medium discriminated resveratrol treatment. ► Resveratrol reduced glycolysis and aminoacid utilisation for energy purpose. ► Mitochondrial succinate utilisation was increased by resveratrol treatment. ►¹³C NMR spectroscopy shows an increase in mitochondrial oleate beta oxidation.

Keywords: Resveratrol; HepG2; Sirt1; Metabolic profiling; NMR spectroscopy; Metabolomics

S-allyl cysteine in combination with clotrimazole downregulates Fas induced apoptotic events in erythrocytes of mice exposed to lead by Samir Mandal; Sudip Mukherjee; Kaustav Dutta Chowdhury; Avik Sarkar; Kankana Basu; Soumosish Paul; Debasish Karmakar; Mahasweta Chatterjee; Tuli Biswas; Gobinda Chandra Sadhukhan; Gargi Sen (pp. 9-23).

Chronic lead (Pb2⁺) exposure leads to the reduced lifespan of erythrocytes. Oxidative stress and K⁺ loss accelerate Fas translocation into lipid raft microdomains inducing Fas mediated death signaling in these erythrocytes. Pathophysiological-based therapeutic strategies to combat against erythrocyte death were evaluated using garlic-derived organosulfur compounds like diallyl disulfide (DADS), S allyl cysteine (SAC) and imidazole based Gardos channel inhibitor clotrimazole (CLT).Morphological alterations in erythrocytes were evaluated using scanning electron microscopy. Events associated with erythrocyte death were evaluated using radio labeled probes, flow cytometry and activity gel assay. Mass spectrometry was used for detection of GSH–4-hydroxy- trans-2-nonenal (HNE) adducts. Fas redistribution into the lipid rafts was studied using immunoblotting technique and confocal microscopy.Combination of SAC and CLT was better than DADS and CLT combination and monotherapy with these agents in prolonging the survival of erythrocytes during chronic Pb2⁺ exposure. Combination therapy with SAC and CLT prevented redistribution of Fas into the lipid rafts of the plasma membrane and downregulated Fas-dependent death events in erythrocytes of mice exposed to Pb2⁺.Ceramide generation was a critical component of Fas receptor-induced apoptosis, since inhibition of acid sphingomyelinase (aSMase) interfered with Fas-induced apoptosis during Pb2⁺ exposure. Combination therapy with SAC and CLT downregulated apoptotic events in erythrocytes by antagonizing oxidative stress and Gardos channel that led to suppression of ceramide-initiated Fas aggregation in lipid rafts. Hence, combination therapy with SAC and CLT may be a potential therapeutic option for enhancing the lifespan of erythrocytes during Pb2⁺ toxicity.► Erythrocyte death during Pb2⁺ exposure is characterized by PS externalization at the outer membrane leaflet. ► Erythrocyte death is triggered by oxidative stress and K⁺ loss. ► The death signaling pathway include redistribution of Fas to lipid rafts and Fas aggregation. ► Combination therapy (S-allyl cysteine and clotrimazole) inhibited ROS generation and K⁺ loss. ► Combination therapy initiated apoptosis inhibiting events in a better way than monotherapy.

Keywords: Abbreviations; FITC; fluorescein isothiocyanate; PS; phosphatidylserine; Prx2; peroxiredoxin2; NAD; nicotinamide adenine dinucleotide; NADP; nicotinamide adenine dinucleotide phosphate; NADH; nicotinamide adenine dinucleotide reduced; NADPH; nicotinamide adenine dinucleotide phosphate reduced; HPF; 3′-(p-hydroxyphenyl) fluorescein; FACS; fluorescence-activated cell sorting; Pb; 2; ⁺; lead; SAC; S allyl cysteine; CLT; clotrimazole; ROS; reactive oxygen species; OH; ⁻; hydroxyl radical; GSH; glutathione; K; ⁺; potassium ion; DMSA; meso-2, 3-dimercaptosuccinic acid; MiADMSA; monoisoamyl meso-2, 3-dimercaptosuccinic acid; DADS; diallyl disulfide; GST; glutathione S transferase; SGPT; serum glutamate pyruvate transaminase; SGOT; serum oxaloacetate transaminase; WBCs; white blood cells; RBCs; red blood cells; PBS; phosphate buffer saline; HNE; hydroxynonenal; TBARS; thiobarbituric acid reactive substance; TBA; thiobarbituric acid; DTNB; 5,5′-dithiobis-2-nitrobenzoic acid; CDNB; 1-cloro-2,4-dinitrobenzene; FCS; fetal calf serum; aSMase; acid sphingomyelinase; BSA; bovine serum albumin; DMTU; dimethyl thiourea; DAS; diallyl sulfide; LDL,; low-density lipoprotein; −; SH group; sulfhydryl group; H; ₂; O; ₂; hydrogen peroxide; GPx; glutathione peroxidase; DISC; death inducing signaling complexLead; Erythrocyte; S allyl cysteine; Clotrimazole; Reactive oxygen species; Apoptosis

Terminal differentiation program of skeletal myogenesis is negatively regulated by O-GlcNAc glycosylation by Mitsutaka Ogawa; Hidenori Mizofuchi; Yuki Kobayashi; Genta Tsuzuki; Mayumi Yamamoto; Shuichi Wada; Kazuo Kamemura (pp. 24-32).

O-Linked β- N-acetylglucosaminylation ( O-GlcNAcylation) on the Ser/Thr residue of nucleocytoplasmic proteins is a dynamic post-translational modification found in multicellular organisms. More than 500 proteins involved in a wide range of cellular functions, including cell cycle, transcription, epigenesis, and glucose sensing, are modified with O-GlcNAc. Although it has been suggested that O-GlcNAcylation is involved in the differentiation of cells in a lineage-specific manner, its role in skeletal myogenesis is unknown.A myogenesis-dependent drastic decrease in the levels of O-GlcNAcylation was found in mouse C2C12 myoblasts. The global decrease in O-GlcNAcylation was observed at the earlier stage of myogenesis, prior to myoblast fusion. Genetic or pharmacological inactivation of O-GlcNAcase blocked both the myogenesis-dependent global decrease in O-GlcNAcylation and myoblast fusion. Although inactivation of O-GlcNAcase affected neither cell-cycle exit nor cell survival in response to myogenic stimulus, it perturbed the expression of myogenic regulatory factors. While the expression of myod and myf5 in response to myogenic induction was not affected, that of myogenin and mrf4 was severely inhibited by the inactivation of O-GlcNAcase.These results indicate that the terminal differentiation program of skeletal myogenesis is negatively regulated by O-GlcNAcylation. O-GlcNAcylation is involved in differentiation in a cell lineage-dependent manner, and a decrease in O-GlcNAcylation may have a common role in the differentiation of cells of muscle lineage.► O-GlcNAcylation decreases drastically during myogenesis in mouse C2C12 myoblasts. ► The decrease in O-GlcNAcylation is involved in myoblast fusion. ► The expression of myogenin and mrf4 is inhibited by the inactivation of O-GlcNAcase.

Keywords: Abbreviations; FBS; fetal bovine serum; HRP; horseradish peroxidase; MHC; myosin heavy chain; MRF; myogenic regulatory factor; O; -GlcNAcylation; O; -linked β-; N; -acetylglucosaminylation; OGT; O; -GlcNAc transferase; PVDF; polyvinylidene difluorideC2C12; Differentiation; Glycosylation; Myoblast; Myogenesis; O; -GlcNAc

Liver X receptor agonist T0901317 induced liver perturbation in zebrafish: Histological, gene set enrichment and expression analyses by Hendrian Sukardi; Xiaoyan Zhang; Eei Yin Lui; Choong Yong Ung; Sinnakaruppan Mathavan; Zhiyuan Gong; Siew Hong Lam (pp. 33-43).

Liver X receptor (LXR), a ligand-activated transcription factor, regulates important biological processes. It has been associated with pathology and proposed as a therapeutic target. The zebrafish is a new vertebrate model for disease modeling, drug and toxicity screening and will be interesting to test for its potential for LXR-related studies.Adult male fish were exposed to LXR agonist T0901317 at 20, 200 and 2000nM for 96h and the livers were sampled for histological, microarray and qRT-PCR analyses.Histological analysis suggests dose-dependent perturbation of carbohydrate and lipid metabolisms by T0901317 in the liver, which lead to hepatocyte swelling and cell death. Microarray data revealed several conserved effects of T0901317 with mammalian models, including up-regulation of LXR-targeted genes, modulation of biological pathways associated with proteasome, cell death, extracellular matrix and adhesions, maturity onset diabetes of the young and lipid beta oxidation. Interestingly, this study identified the complement and coagulation systems as down-regulated by T0901317 for the first time, potentially via transcriptional repression by LXR activation. qRT-PCR validated the expression of 16 representative genes, confirming activation of LXR signaling and down-regulation of these biological pathways by T0901317 which could be linked to the anti-thrombogenic, anti-atherogenic and anti-inflammatory actions, as well as metabolic disruptions via LXR activation.Our study underscores the potential of using zebrafish model coupled with transcriptomic analysis to capture pharmacological and toxicological or pathological events induced by LXR modulators.► Zebrafish treated with LXR agonist T0901317 were analyzed by microarray and histology. ► T0901317 perturbed carbohydrate and lipid metabolism with swelling hepatocytes. ► Effects of T0901317 on LXR modulated pathways were conserved in zebrafish and mammals. ► Microarray analysis identified complement and coagulation systems as novel LXR targets. ► PCR validation confirmed T0901317 induced LXR signaling and pharmacological effects.

Keywords: Zebrafish; Liver X receptor; T0901317; Liver; Toxicogenomics; Transcriptome

Phosphorylation of the arginine/serine repeats of lamin B receptor by SRPK1—Insights from molecular dynamics simulations by Diamantis Sellis; Victoria Drosou; Dimitrios Vlachakis; Nikolas Voukkalis; Thomas Giannakouros; Metaxia Vlassi (pp. 44-55).

Arginine/serine (RS) repeats are found in several proteins in metazoans with a wide variety of functions, many of which are regulated by SR protein kinase 1 (SRPK1)-mediated phosphorylation. Lamin B receptor (LBR) is such a protein implicated in chromatin anchorage to the nuclear envelope.Molecular dynamics simulations were used to investigate the conformation of two LBR peptides containing four (human-) and five (turkey-orthologue) consecutive RS dipeptides, in their unphosphorylated and phosphorylated forms and of a conserved peptide, in isolation and in complex with SRPK1. GST pull-down assays were employed to study LBR interactions.Unphosphorylated RS repeats adopt short, transient helical conformations, whereas serine phosphorylation induces Arginine-claw-like structures. The SRSRSRSPGR peptide, overlapping with the LBR RS repeats, docks into the known, acidic docking groove of SRPK1, in an extended conformation. Phosphorylation by SRPK1 is necessary for the association of LBR with histone H3.The C-terminal region of the LBR RS domain constitutes a recognition platform for SRPK1, which uses the same recognition mechanism for LBR as for substrates with long RS domains. This docking may promote unfolding of the RS repeats destined to be phosphorylated. Phosphorylation induces Arginine-claw-like conformations, irrespective of the RS-repeat length, that may facilitate interactions with basic partners.Our results shed light on the conformational preferences of an important class of repeats before and after their phosphorylation and support the idea that even short RS domains may be constituents of recognition platforms for SRPK1, thus adding to knowledge towards a full understanding of their phosphorylation mechanism.► The unphosphorylated RS repeats of LBR adopt short transient helical structures. ► Phosphorylation of RS repeats induces the formation of Arg-claw-like structures. ► The RSRSPGR peptide of LBR constitutes the docking motif for SRPK1. ► Part of short RS domains may be constituents of docking motifs for SRPK1. ► The docking interactions with SRPK1 may promote unfolding of the RS repeats of LBR.

Keywords: RS repeats; Lamin B receptor; Phosphorylation; SRPK1; Molecular dynamics simulations

Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance by Mohsen Rajabi; Evi Struble; Zhaohua Zhou; Elena Karnaukhova (pp. 56-63).

Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states.We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout.Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation.This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies.► A novel (consecutive double capture) SPR approach. ► A significant acceleration of the C1-INH interactions with C1s due to heparin. ► Conformational assessment. ► Functional assay. ► Serpin structural overview and heparin binding sites.

Keywords: Abbreviations; Anti-C1-INH ab; anti-C1-esterase inhibitor antibody; α; ₁; -PI; α; ₁; -proteinase inhibitor; ATIII; antithrombin; C1-INH; C1-esterase inhibitor; CD; circular dichroism; DMSO; dimethyl sulfoxide; GAG; glycosaminoglycan; L/P; ligand-to-protein molar ratio; PBS; phosphate buffer saline; RCL; reactive center loop; SA; Streptavidin; SPR; Surface Plasmon ResonanceC1-esterase inhibitor; C1s; Heparin; Surface plasmon resonance

Sections

Personal tools

BBA - General Subjects (v.1820, #1)

Sections

Personal tools

Local resources

BBA - General Subjects (v.1820, #1)