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BBA - General Subjects (v.1830, #1)

Editorial Board (pp. i).

Treatment with pharmacological PPARα agonists stimulates the ubiquitin proteasome pathway and myofibrillar protein breakdown in skeletal muscle of rodents by Robert Ringseis; Janine Keller; Isabel Lukas; Julia Spielmann; Erika Most; Aline Couturier; Konig Bettina König; Frank Hirche; Gabriele I. Stangl; Gaiping Wen; Klaus Eder (pp. 2105-2117).

Treatment of hyperlipidemic patients with fibrates, agonists of peroxisome proliferator-activated receptor α (PPARα), provokes muscle atrophy as a side effect. The molecular mechanism underlying this phenomenon is still unknown. We tested the hypothesis that activation of PPARα leads to an up-regulation of the ubiquitin proteasome system (UPS) which plays a major role in protein degradation in muscle.Rats, wild-type and PPARα-deficient mice (PPARα^−/−) were treated with synthetic PPARα agonists (clofibrate, WY-14,643) to study their effect on the UPS and myofibrillar protein breakdown in muscle.In rats and wild-type mice but not PPARα^−/− mice, clofibrate or WY-14,643 caused increases in mRNA and protein levels of the ubiquitin ligases atrogin-1 and MuRF1 in muscle. Wild-type mice treated with WY-14,643 had a greater 3-methylhistidine release from incubated muscle and lesser muscle weights. In addition, wild-type mice but not PPARα^−/− mice treated with WY-14,643 had higher amounts of ubiquitin–protein conjugates, a decreased activity of PI3K/Akt1 signalling, and an increased activity of FoxO1 transcription factor in muscle. Reporter gene and gel shift experiments revealed that the atrogin-1 and MuRF1 promoter do not contain functional PPARα DNA-binding sites.These findings indicate that fibrates stimulate ubiquitination of proteins in skeletal muscle which in turn stimulates protein degradation. Up-regulation of ubiquitin ligases is probably not mediated by PPARα-dependent gene transcription but by PPARα-dependent inhibition of the PI3K/Akt1 signalling pathway leading to activation of FoxO1.PPARα plays a role in the regulation of the ubiquitin proteasome system.► PPARα activators increase expression of ubiquitin ligases in muscle of rodents. ► PPARα activators enhance 3-methylhistidine release from incubated muscle in mice. ► PPARα activators decrease muscle weights in mice. ► PPARα activators increase amounts of ubiquitin–protein conjugates in muscle of mice. ► PPARα activators reduce activity of PI3K/Akt1 signalling in muscle of mice.

Keywords: Abbreviations; ACOX; acyl-CoA oxidase; ChIP; chromatin immunoprecipitation; CPT; carnitine-palmitoyltransferase; CTSL; L-cathepsin; DTT; dithiothreitol; EDL; extensor digitorum longus; FoxO1; forkhead box protein O1; 3-MH; 3-methyl histidine; MuRF1; muscle RING finger-1; PI3K; phosphatidylinositol 3-kinase; PPARα; peroxisome proliferator-activated receptor α; PPRE; peroxisome proliferator response element; RXRα; retinoid X receptor α; UPS; ubiquitin proteasome systemPeroxisome proliferator-activated receptor α; Ubiquitin proteasome system; Atrogin-1; MuRF1; Muscle atrophy

Implication of intestinal VDR deficiency in inflammatory bowel disease by Jung-Hwan Kim; Satoshi Yamaori; Tomotaka Tanabe; Caroline H. Johnson; Kristopher W. Krausz; Shigeaki Kato; Frank J. Gonzalez (pp. 2118-2128).

To investigate the function of the intestinal Vdr gene in inflammatory bowel disease (IBD), in conjunction with the discovery of possible metabolic markers for IBD using intestine-specific Vdr knockout mice. Vdr^ΔIEpC mice were generated, phenotyped and treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis. Colitis was diagnosed by evaluating clinical symptoms and intestinal histopathology. Gene expression analysis was carried out. In addition, metabolic markers of IBD were explored by metabolomics. Vdr^ΔIEpC mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. In the acute model which was treated with 3% DSS for six days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; enlarged mucosal layer and damaged muscularis layer. In the recovery experiment model, where mice were treated with 3% DSS for four days and water for three days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; severe damage of mucosal layer and increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in Vdr^ΔIEpC mice.Disruption of the intestinal Vdr gene showed phenotypical changes that may exacerbate IBD. These results suggest that VDR may play an important role in IBD. General significanceVDR function has been implicated in IBD. This is of value for understanding the etiology of IBD and for development of diagnostic biomarkers for IBD.► VdrΔIEpC mice were generated and showed changes in body mass, blood chemistry, colon structure, gene expression profile and the fecal metabolome ► The DSS-induced colitis model was used in the VdrΔIEpC mice and showed susceptibility to IBD ► Taurine and taurine conjugated bile acids could be diagnostic biomarkers for intestinal diseases.

Keywords: Abbreviations; VDR; vitamin D receptor; IBD; inflammatory bowel disease; ALP; alkaline phosphatase; ALT; alanine aminotransferase; qPCR; quantitative polymerase chain reaction; UPLC–ESI–QTOFMS; ultra-performance liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry; DSS; dextran sulfate sodium; CHL; cholesterol; TBIL; total bilirubin; PCNA; proliferating cell nuclear antigen; LPS; lipopolysaccharides; FXR; farnesoid X receptor; H&E; hematoxylin and eosinVitamin D receptor (VDR); Inflammatory bowel disease (IBD); Bile acid

Ligand and pathogen specificity of the Atlantic salmon serum C-type lectin by Elke Uribe; Thomas J. Steele; Robert C. Richards; K. Vanya Ewart (pp. 2129-2138).

An Atlantic salmon ( Salmo salar) C-type lectin (SSL) binds to mannose and related sugars as well as to the surface of Aeromonas salmonicida. To characterize this lectin as a pathogen recognition receptor in salmon, aspects of its interaction with molecules and with intact pathogens were investigated.SSL was isolated using whole-yeast-affinity and mannan-affinity chromatography. The binding of SSL to the two major surface molecules of A. salmonicida, lipopolysaccharide (LPS) and A-layer protein was investigated by western blotting and enzyme-linked immunosorbent assays. Microbial binding specificity of SSL was examined by whole cell binding assays using a range of species. Carbohydrate ligand specificity of SSL was examined using glycan array analysis and frontal affinity chromatography.SSL showed binding to bacteria and yeast including, Pseudomonas fluorescens, A. salmonicida, A. hydrophila, Pichia pastoris, and Saccharomyces cerevisiae, but there was no detectable binding to Yersinia ruckeri. In antimicrobial assays, SSL showed no activity against Escherichia coli, Bacillus subtilis, S. cerevisiae, or A. salmonicida, but it was found to agglutinate E. coli. The major surface molecule of A. salmonicida recognized by SSL was shown to be LPS and not the A-layer protein. LPS binding was mannose-inhibitable. Glycans containing N-acetylglucosamine were shown to be predominant ligands.SSL has a distinct ligand preference while allowing recognition of a wide variety of related carbohydrate structures.SSL is likely to function as a wide-spectrum pattern recognition protein.► Atlantic salmon serum lectin (SSL) recognizes specific bacteria and fungi. ► Laboratory analyses reveal SSL binding to A. salmonicida lipopolysaccharide. ► Glycan array-binding experiments show SSL binding to glycans with terminal N-acetylglucosamine.

Keywords: Atlantic salmon; Salmo salar; C-type lectin; Glycan array; Lipopolysaccharide; Pathogen recognition

Zinc induced folding is essential for TIM15 activity as an mtHsp70 chaperone by Hugo Fraga; Elena Papaleo; Sonia Vega; Adrián Velazquez-Campoy; Salvador Ventura (pp. 2139-2149).

TIM15/Zim17 in yeast and its mammalian ortholog Hep are Zn2⁺ finger (Cys4) proteins that assist mtHsp70 in protein import into the mitochondrial matrix.Here we characterized the Zn2⁺ induced TIM15 folding integrating biophysical and computational approaches.TIM15 folding occurs from an essentially unstructured conformation to a Zn2⁺-coordinated protein in a fast and markedly temperature-dependent process. Moreover, we demonstrate unambiguously that Zn2⁺ induced TIM15 folding is essential for its role as mtHsp70 chaperone since in the unstructured apo state TIM15 does not bind to mtHsp70 and is unable to prevent its aggregation. Molecular dynamics simulations help to understand the crucial role of Zn2⁺ in promoting a stable and functional 3D architecture in TIM15. It is shown that the metal ion, through its coordinating cysteine residues, can mediate relevant long-range effects with the interaction interface for mtHsp70 coupling thus folding and function.Zn2⁺ induced TIM15 folding is essential for its function and likely occurs in mitochondrial matrix where high concentrations of Zn2⁺ were reported.The combination of experimental and computational approaches presented here provide an integrated structural, kinetic and thermodynamic view of the folding of a mitochondrial zinc finger protein, which might be relevant to understand the organelle import of proteins sharing this fold.Display Omitted► TIM15 is an essential Zn2⁺ finger mitochondrial protein. ► A combined biophysical and molecular dynamics characterization of TIM15 folding. ► TIM15 folding and function depend on Zn2⁺. ► TIM15 Zn2⁺ induced folding is a fast and markedly temperature-dependent process. ► TIM15 Zn2⁺ induced folding is essential for its role as mtHsp70 chaperone.

Keywords: Abbreviations; AMS; (4-acetamido-49-maleimidylstibene-2,29-disulfonic acid); ANS; 8-anilino-1-naphthalenesulfonic acid; DCCM; dynamical cross-correlation matrix; DSC; differential scanning calorimetry; DTT; dithiothreitol; EDTA; ethylene diamine tetraacetic acid; ITC; isothermal titration calorimetry; MD; molecular dynamics; PSN; protein structure network; TCEP; tris(2-carboxyethyl)phosphine; ThT; thioflavin TTIM15; Mitochondrion; Protein folding; Zinc finger; Protein translocation

Optimized negative-staining electron microscopy for lipoprotein studies by Lei Zhang; Huimin Tong; Mark Garewal; Gang Ren (pp. 2150-2159).

Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents.The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed.The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1nm, but rarely better than 1nm) method which has been used to discover the mechanics of a small protein, 53kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14Å-resolution IgG antibody three-dimensional map.It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures.► Negative-staining (NS) EM is a rapid, common method to examine protein structure. ► We reviewed the most popular NS protocols used to examine lipoproteins. ► The rouleau artifact of lipoproteins was commonly observed using conventional NS protocol. ► The optimized NS (OpNS) protocol can eliminate these rouleau artifacts. ► OpNS can be used as a general protocol to study the structure of proteins.

Keywords: Abbreviations; EM; electron microscopy; NS; negative staining; OpNS; optimized negative staining; cryo-EM; cryo-electron microscopy; IPET; individual-particle electron tomography; HDL; high-density lipoprotein; LDL; low-density lipoprotein; VLDL; very low-density lipoprotein; IDL; intermediate-density lipoprotein; UF; uranyl formate; PTA; phosphotungstic acid; SNR; signal-to-noise ratio; CVD; cardiovascular diseaseProtein structure; Lipoprotein structure; Electron microscopy; Negative-staining; Optimized negative-staining protocol; Individual-particle electron tomography

Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation by Sola Susana Solá; Ana L. Morgado; Cecília M.P. Rodrigues (pp. 2160-2166).

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.► Cell death-relevant proteins are functionally involved in neural differentiation. ► Understanding differentiation will improve neuro-replacement strategies. ► Mitochondrial and death receptor pathways influence stem cell fate. ► Neuronal plasticity and astrocytic versus neuronal fate decision are also modulated. ► Cell fate switch involves regulation of redox stage and Bcl-2 family proteins.

Keywords: Abbreviations; Apaf-1; apoptosis protease activating factor-1; Bcl-2; B-cell lymphoma-2; CNS; central nervous system; ESCs; embryonic stem cells; IAPs; inhibitor of apoptosis proteins; IL-1α/β; interleukin-1 α/β; iPSCs; induced pluripotent stem cells; miRNAs; microRNAs; MMP; mitochondrial membrane permeabilization; MSCs; mesenchymal stem cells; mtDNA; mitochondrial DNA; NPCs; neural progenitor cells; NSCs; neural stem cells; NF-κB; nuclear factor-κB; ROS; reactive oxygen species; SVZ; subventricular zone; TNF; tumor necrosis factor; TNF-α; TNF ligand α; TNFR1/2; TNF-receptor type 1/2Death receptor; Mitochondrion; Neural differentiation; Stem cell

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis by Xavier Roussel; Christine Lancelon-Pin; Anders Viksø-Nielsen; Rolland-Sabate Agnès Rolland-Sabaté; Florent Grimaud; Potocki-Veronese Gabrielle Potocki-Véronèse; Buleon Alain Buléon; Jean-Luc Putaux; Christophe D'Hulst (pp. 2167-2177).

Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch.The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure.A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3–8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30–70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family.RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family.This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.► Branching enzyme of R. obamensis (RoBE) requires minimal DP10 donor glucans. ► RoBE produces highly branched polymers from a mix of polydisperse linear glucans. ► The branching process is preferably intermolecular. ► Branched polymers are preferred acceptor substrates. ► RoBE has an extra α-4-glucanotransferase activity not reported yet for such enzyme.

Keywords: Branching enzyme; Rhodothermus obamensis; Glycogen; Dextrin; Polyglucan; In vitro synthesis

Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis by Jia-Hsin Huang; Jesus Lozano; Xavier Belles (pp. 2178-2187).

Insect metamorphosis proceeds in two modes: hemimetaboly, gradual change along the life cycle; and holometaboly, abrupt change from larvae to adult mediated by a pupal stage. Both are regulated by 20-hydroxyecdysone (20E), which promotes molts, and juvenile hormone (JH), which represses adult morphogenesis. Expression of Broad-complex (BR-C) is induced by 20E and modulated by JH. In holometabolous species, like Drosophila melanogaster, BR-C expression is inhibited by JH in young larvae and enhanced in mature larvae, when JH declines and BR-C expression specifies the pupal stage.Using Blattella germanica as a basal hemimetabolous model, we determined the patterns of expression of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA interference approaches.We found that BR-C expression is enhanced by JH and correlates with JH hemolymph concentration. BR-C factors appear to be involved in cell division and wing pad growth, as well as wing vein patterning.In B. germanica, expression of BR-C is enhanced by JH, and BR-C factors appear to promote wing growth to reach the right size, form and patterning, which contrast with the endocrine regulation and complex functions observed in holometabolous species.Our results shed new light to the evolution from hemimetaboly to holometaboly regarding BR-C, whose regulation and functions were affected by two innovations: 1) a shift in JH action on BR-C expression during young stages, from stimulatory to inhibitory, and 2) an expansion of functions, from regulating wing development, to determining pupal morphogenesis.► In cockroaches, juvenile hormone induces the expression of Broad-complex in young stages. ► In flies, juvenile hormone inhibits its expression of Broad-complex in young stages. ► In cockroaches, Broad-complex promotes wing growth and patterning. ► In flies, it specifies pupal morphogenesis. ► Broad-complex played a key role in metamorphosis evolution from cockroaches to flies.

Keywords: Insect metamorphosis; Juvenile hormone; Ecdysone; Evolution of holometaboly; Drosophila; Tribolium

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion by Saskia A. Overbeek; Marije Kleinjan; Paul A.J. Henricks; Vera M. Kamp; Fabio L. Ricciardolo; Niki A. Georgiou; Johan Garssen; Aletta D. Kraneveld; Gert Folkerts (pp. 2188-2193).

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.► We investigate the effect of N-ac-PGP on neutrophilic adhesion using fibrinogen. ► To determine the contribution of Mac-1 to neutrophilic adhesion, antibodies were used. ► We examine the effect of N-ac-PGP on the Mac-1 expression pattern. ► N-ac-PGP induces a conformational change of Mac-1 leading to neutrophilic adhesion.

Keywords: Abbreviations; COPD; chronic obstructive pulmonary disease; FBS; fetal bovine serum; fMLP; formyl-Methionyl-Leucyl-Phenylalanine; GPCR; G protein-coupled receptor; HUVEC; human umbilical vein endothelial cell; LFA-1; lymphocyte Function-associated Antigen 1, CD11a/CD18, α; _L; β; ₂; MMP; matrix metalloprotease; PE; prolyl endopeptidase; Mac-1; macrophage 1 antigen, CD11b/CD18, α; _M; β; ₂; N-ac-PGP; N-acetylated Proline–Glycine–Proline; PBS; phosphate buffered saline; PMN; polymorphonuclear leukocyte; PTX; pertussis toxin; RPMI; Roswell Park Memorial InstitutePolymorphonuclear leukocytes; Collagen breakdown; β; ₂; -Integrin; Chronic obstructive pulmonary disease

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, inhibits human oral squamous carcinoma cells KB and KB/VCR: In vitro and in vivo studies by Bin Yue; Cui-Rong Zhao; Hui-Min Xu; Yuan-Yuan Li; Yan-Na Cheng; Han-Ni Ke; Yi Yuan; Rui-Qi Wang; Yan-Qiu Shi; Hong-Xiang Lou; Xian-Jun Qu (pp. 2194-2203).

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, might possess anti-cancer properties. We aimed to evaluate the efficacy of Riccardin D-26 as a candidate compound for treatment of cancers with sensitive or drug resistant cells.Experiments were performed on human oral squamous carcinoma KB cells and vincristin-selected MDR KB/VCR cells. The inhibition of cell growth was evaluated by colorimetric and clonogenic assays. The apoptotic cells were determined by the Annexin V-FITC/PI staining assay. JC-1 fluorescence probe was used to examine the mitochondria membrane potential (MMP). Further experiments were performed in nude mice bearing KB or KB/VCR xenografts. Riccardin D-26 was administered by injection for 2weeks. The specimens of KB and KB/VCR xenografts were removed for TUNEL staining and Western blotting analysis.Riccardin D-26 significantly inhibited cancer growth in both KB and KB/VCR cells. Riccardin D-26's activity in cancer cells was greater than that in human normal liver cells. In mice, Riccardin D-26 effectively prevented the growth of KB and KB/VCR xenografts without significant toxicity. Further studies suggested that Riccardin D-26 inhibited cancer growth by inducing apoptosis in the activation of mitochondria-mediated intrinsic apoptosis pathway. Riccardin D-26 also possessed this activity in regulation of mitogen-related protein kinases such as MAPK and PI3K/Akt, which is associated with its inhibitory effect on KB/VCR cells.Riccardin D-26 possessed an anti-proliferation activity against both sensitive KB and MDR KB/VCR cancer cells.Riccardin D-26 could be a promising agent for treatment of cancers with sensitive or drug resistant cells.► Riccardin D-26 is a derivative of the synthesized macrocyclic bisbibenzyl compound. ► Riccardin D-26 possessed activity against both KB and KB/VCR cells. ► Riccardin D-26 inhibited cancer growth in mice with no significant toxicity. ► The effect of Riccardin D-26 on KB/VCR cells was due to induction of apoptosis. ► Riccardin D-26 possessed the activity in regulation of MAPK and PI3K/Akt.

Keywords: Synthesized macrocyclic bisbibenzyl compound; KB/VCR; MDR; Mitochondria-mediated intrinsic apoptosis pathway; MAPK; PI3K/Akt

CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases by Naoyuki Okita; Miyuki Yoshimura; Kazuhito Watanabe; Shota Minato; Yuki Kudo; Yoshikazu Higami; Sei-ichi Tanuma (pp. 2204-2213).

CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles.In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at²⁹⁶SNLD²⁹⁹ and³⁴⁸TCPD³⁵¹, and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified³²⁰EPRT³²³ as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at³²⁰EPRT³²³. Additionally, the cleavage catalyzed by the³²⁰EPRT³²³ protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment.CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the “³²⁰EPRT³²³ protease(s).” Furthermore,³²⁰EPRT³²³ cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage.CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.► CHK1 is an important effector kinase that regulates the cell cycle checkpoint. ► CHK1 is cleaved at three sites in programmed cell death. ► Serine protease(s) is involved in CHK1 cleavage. ► CHK1 is cleaved by both CASP and non-CASP family proteases. ► CHK1 cleavage results in the enhancement of CHK1 kinase activity.

Keywords: CHK1; DNA damage response; Programmed cell death; Caspase family proteases; Non-caspase family proteases

Diallyl tetrasulfane activates both the eIF2α and Nrf2/HO-1 pathways by Nathaniel Edward Bennett Saidu; Rania Touma; Imad Abu Asali; Claus Jacob; Mathias Montenarh (pp. 2214-2225).

Diallyl polysulfanes have been shown to exert cell cycle arrest, anti-tumor and anti-inflammatory activities in a variety of in vitro and in vivo models. Although diallyl polysulfanes cause oxidative stress, little is known about the underlying signaling cascades leading to antioxidant defense or apoptosis.Cells were treated with DATTS at different concentrations and for different time periods. Reactive oxygen species and thiol concentrations were determined by commercially available kits. The expression levels of signal molecules were determined by Western Blot analysis. A direct influence of Nrf2 on the promoter of HO-1 gene was determined by a luciferase assay with the StRE promoter element from the HO-1 gene.We found an immediate increase in the level of the superoxide anion radical O₂⁻ and hydrogen peroxide H₂O₂ and an overall thiol depletion. DATTS treatment of HCT116 cells also caused an up-regulation of phospho-eIF2α, nuclear Nrf2 and HO-1 protein levels in a time and concentration-dependent manner. Pre-treatment of cells with antioxidants significantly reduced the elevated expression levels of these proteins. A direct contribution of Nrf2 was shown by its interaction with the stress–response element of the HO-1 promoter.DATTS activates the ROS-eIF2α/Nrf2 HO-1 signaling cascades leading to the up-regulation of HO-1. However, this antioxidant defense is not sufficient to protect HCT116 cells from apoptosis.This study shows for the first time a parallel but not equal activation of signaling pathways by DATTS with a competitive ultimate cellular outcome.Display Omitted► Diallyl tetrasulfane (DATTS) causes an immediate increase in reactive oxygen species. ► DATTS treatment of cells leads to an immediate decrease in total thiol level. ► DATTS induces signaling pathways leading to an antioxidant response and/or apoptosis. ► A competition between antioxidant defense and apoptosis determines the cellular outcome.

Keywords: Reactive oxygen species (ROS); Thiol; Redox reagent; Antioxidant pathway; Apoptosis

Lipoic acid prevents liver metabolic changes induced by administration of a fructose-rich diet by María C. Castro; María L. Massa; Guillermo Schinella; Juan J. Gagliardino; Flavio Francini (pp. 2226-2232).

To evaluate whether co-administration of R/S-α-lipoic acid can prevent the development of oxidative stress and metabolic changes induced by a fructose-rich diet (F).We assessed glycemia in the fasting state and during an oral glucose tolerance test, triglyceridemia and insulinemia in rats fed with standard diet (control) and fructose without or with R/S-α-lipoic acid. Insulin resistance and hepatic insulin sensitivity were also calculated. In liver, we measured reduced glutathione, protein carbonyl groups, antioxidant capacity by ABTS assay, antioxidant enzymes (catalase and superoxide dismutase 1 and 2), uncoupling protein 2, PPARδ and PPARγ protein expressions, SREBP-1c, fatty acid synthase and glycerol-3-phosphate acyltransferase-1 gene expression, and glucokinase activity.R/S-α-lipoic acid co-administration to F-fed rats a) prevented hyperinsulinemia, hypertriglyceridemia and insulin resistance, b) improved hepatic insulin sensitivity and glucose tolerance, c) decreased liver oxidative stress and increased antioxidant capacity and antioxidant enzymes expression, d) decreased uncoupling protein 2 and PPARδ protein expression and increased PPARγ levels, e) restored the basal gene expression of PPARδ, SREBP-1c and the lipogenic genes fatty acid synthase and glycerol-3-phosphate acyltransferase, and f) decreased the fructose-mediated enhancement of glucokinase activity.Our results suggest that fructose-induced oxidative stress is an early phenomenon associated with compensatory hepatic metabolic mechanisms, and that treatment with an antioxidant prevented the development of such changes.This knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced oxidative stress and to develop effective strategies to prevent and treat, at early stages, obesity and type 2 diabetes mellitus.► Fructose-induced oxidative stress is an early phenomenon related to compensatory hepatic metabolic mechanisms. ► Modified PPARδ, PPARγ and UCP2 expressions might be hepatic adaptations to fructose-induced oxidative stress. ► Effective prevention of the oxidative stress by lipoic acid suggest its potential clinical treatment value. ► Our data would help to develop effective strategies to prevent and treat, at early stages, obesity and type 2 diabetes.

Keywords: Abbreviations; T2DM; type 2 diabetes mellitus; F; fructose-rich diet; ROS; reactive oxygen species; UCP; uncoupling protein; LA; R/S-α-lipoic acid; FAS; fatty acid synthase; GPAT-1; glycerol-3-phosphate acyltransferase-1; HOMA-IR; homeostasis model assessment-insulin resistance; FPI; fasting plasma insulin; FPG; fasting plasma glucose; OGTT; oral glucose tolerance test; AUC; area under the glucose curve; GSH; reduced glutathione; SOD1; superoxide dismutase 1; SOD2; superoxide dismutase 2; ABTS; 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)Fructose; Liver uncoupling protein 2; PPAR regulation; Glycoxidative stress; R/S-α-lipoic acid; Pre-diabetes

Cis-9,trans-11-conjugated linoleic acid affects lipid raft composition and sensitizes human colorectal adenocarcinoma HT-29 cells to X-radiation by Gradzka Iwona Grądzka; Barbara Sochanowicz; Brzoska Kamil Brzóska; Wojciuk Grzegorz Wójciuk; Sylwester Sommer; Wojewodzka Maria Wojewódzka; Gasinska Anna Gasińska; Christian Degen; Gerhard Jahreis; Irena Szumiel (pp. 2233-2242).

Investigations concerned the mechanism of HT-29 cells radiosensitization by cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA), a natural component of human diet with proven antitumor activity.The cells were incubated for 24h with 70μM c9,t11-CLA and then X-irradiated. The following methods were used: gas chromatography (incorporation of the CLA isomer), flow cytometry (cell cycle), cloning (survival), Western blotting (protein distribution in membrane fractions), and pulse-field gel electrophoresis (rejoining of DNA double-strand breaks). In parallel, DNA-PK activity, γ-H2AX foci numbers and chromatid fragmentation were estimated. Gene expression was analysed by RT-PCR and chromosomal aberrations by the mFISH method. Nuclear accumulation of the EGF receptor (EGFR) was monitored by ELISA.C9,t11-CLA sensitized HT-29 cells to X-radiation. This effect was not due to changes in cell cycle progression or DNA-repair-related gene expression. Post-irradiation DSB rejoining was delayed, corresponding with the insufficient DNA-PK activation, although chromosomal aberration frequencies did not increase. Distributions of cholesterol and caveolin-1 in cellular membrane fractions changed. The nuclear EGFR translocation, necessary to increase the DNA-PK activity in response to oxidative stress, was blocked. We suppose that c9,t11-CLA modified the membrane structure, thus disturbing the intracellular EGFR transport and the EGFR-dependent pro-survival signalling, both functionally associated with lipid raft properties.The results point to the importance of the cell membrane interactions with the nucleus after injury inflicted by X -rays. Compounds like c9,t11-CLA, that specifically alter membrane properties, could be used to develop new anticancer strategies.► Mechanism of radiosensitization by c9,t11-CLA was studied in HT-29 cells. ► C9,t11-CLA supplementation affects the cholesterol and caveolin distribution in lipid rafts. ► X-irradiation-induced translocation of EGFR to the cell nuclei is inhibited. ► DNA-PK activity is not sufficiently stimulated in response to X-irradiation. ► Rejoining of double-strand breaks in DNA is disturbed.

Keywords: Abbreviations; CLA; conjugated linoleic acid(s); DNA-PK; DNA-dependent protein kinase; DSB; DNA double-strand break; EGFR; epidermal growth factor receptor; FAME; fatty acid methyl ester(s); GC; gas chromatography; LA; linoleic acid; mFISH; multicolour fluorescence in situ hybridization; MUFA; monounsaturated fatty acid(s), non-esterified fatty acids; PCC; premature chromosome condensation; PFGE; pulse-field gel electrophoresis; PL; phospholipid(s); RT-PCR; real-time polymerase chain reaction; SFA; saturated fatty acid(s); TAG; triacylglycerol(s); TLC; thin-layer chromatographyCis-9,trans-11-conjugated linoleic acid; HT-29 cells; Radiosensitization; Lipid raft; EGF receptor; DNA double-strand break

Insulin sensitization via partial agonism of PPARγ and glucose uptake through translocation and activation of GLUT4 in PI3K/p-Akt signaling pathway by embelin in type 2 diabetic rats by Gopalsamy Rajiv Gandhi; Antony Stalin; Kedike Balakrishna; Savarimuthu Ignacimuthu; Michael Gabriel Paulraj; Rajagopal Vishal (pp. 2243-2255).

The present study was aimed at isolating an antidiabetic molecule from a herbal source and assessing its mechanism of action.Embelin, isolated from Embelia ribes Burm. (Myrsinaceae) fruit, was evaluated for its potential to regulate insulin resistance, alter β-cell dysfunction and modulate key markers involved in insulin sensitivity and glucose transport using high-fat diet (HFD) fed-streptozotocin (STZ) (40mg/kg)-induced type 2 diabetic rats. Molecular-dockings were performed to investigate the binding modes of embelin into PPAR γ, PI3K, p-Akt and GLUT4 active sites.Embelin (50mg/kg b wt.) reduced body weight gain, blood glucose and plasma insulin in treated diabetic rats. It further modulated the altered lipid profiles and antioxidant enzymes with cytoprotective action on β-cell. Embelin significantly increased the PPARγ expression in epididymal adipose tissue compared to diabetic control group; it also inhibited adipogenic activity; it mildly activated PPAR γ levels in the liver and skeletal muscle. It also regulated insulin mediated glucose uptake in epididymal adipose tissue through translocation and activation of GLUT4 in PI3K/p-Akt signaling cascade. Embelin bound to PPARγ; it disclosed stable binding affinities to the active sites of PI3K, p-Akt and GLUT4.These findings show that embelin could improve adipose tissue insulin sensitivity without increasing weight gain, enhance glycemic control, protect β-cell from damage and maintain glucose homeostasis in adipose tissue.Embelin can be used in the prevention and treatment of type 2 diabetes mellitus caused due to obesity.Display Omitted► Embelin was isolated from Embelia ribes Burm. fruit. ► Embelin suppressed the body weight gain in type 2 diabetic rats. ► Embelin increased the PPARγ expression in epididymal adipose tissue. ► Embelin regulated glucose uptake through GLUT4 in PI3K/ p-Akt signaling. ► Embelin docked with PPARγ, PI3K, p-Akt and GLUT4.

Keywords: Abbreviations; T2DM; Type 2 diabetes mellitus; HFD; high fat diet; STZ; streptozotocin; TZDs; Thiazolidinediones; FBG; fasting blood glucose; b wt.; body weight; TC; total cholesterol; TG; triglycerides; FFA; free fatty acids; SOD; superoxide dismutase; CAT; catalase; GPx; glutathione peroxidase; OGTT; oral glucose tolerance test; PBS; phosphate buffer saline; MW; Molecular weightEmbelin; Type 2 diabetes mellitus; PPARγ; GLUT4; PI3K/p-Akt; Molecular-docking

Cellular internalization and stress response of ingested amorphous silica nanoparticles in the midgut of Drosophila melanogaster by Ashutosh Pandey; Swati Chandra; Lalit Kumar Singh Chauhan; Gopeshwar Narayan; Debapratim Kar Chowdhuri (pp. 2256-2266).

Amorphous silica nanoparticles (aSNPs) are used for various applications including food industry. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the midgut cells of an organism. The study aims to examine cellular uptake of aSNPs (<30nm) in the midgut of Drosophila melanogaster (Oregon R⁺) owing to similarities between the midgut tissue of this organism and human and subsequently cellular stress response generated by these nanoparticles.Third instar larvae of D. melanogaster were exposed orally to 1–100μg/mL of aSNPs for 12–36h and oxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bromide staining), cellular internalization (TEM) and apoptosis endpoints.A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in a concentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 along with caspases activation, membrane destabilization and mitochondrial membrane potential loss was also observed. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles and by direct membrane penetration.aSNPs after their internalization in the midgut cells of exposed Drosophila larvae show membrane destabilization along with increased cellular stress and cell death.Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thus their industrial use as a food-additive may raise concern to human health.► Cellular internalization of ingested aSNPs by endocytosis and direct penetration ► aSNPs mediated membrane destabilization in the midgut cells of Drosophila. ► aSNPs increased ROS generation and evoked oxidative stress in midgut cells. ► aSNPs induced heat shock genes ( hsp70, hsp22) in midgut cells. ► aSNPs induced mitochondrial membrane potential loss and apoptosis in midgut cells.

Keywords: Abbreviations; aSNPs; Amorphous silica nanoparticles; NMs; Nanomaterials; NPs; Nanoparticles; ENPs; Engineered nanoparticles; GI; Gastrointestinal; Hsps; Heat shock proteins; Hsgs; Heat shock genes; Sps; Stress proteins; sHsps; Small heat shock proteins; OS; Oxidative stress; ROS; Reactive oxygen species; SOD; Superoxide dismutase; CAT; Catalase; LPO; Lipid peroxidation; GSH; Glutathione; GST; Glutathione S-transferase; PC; Protein content; DNPH; Di-nitro phenyl hydrazine; PBS; Phosphate buffered saline; BSA; Bovine serum albumin; TEM; Transmission electron microscopy; DLS; Dynamic light scattering; AO/EB; Acridine orange/Ethidium Bromide; MMP; Mitochondrial membrane potentialAmorphous silica nanoparticles; Heat shock proteins; Oxidative stress; Membrane destabilization; Caspase-3; Cellular uptake

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BBA - General Subjects (v.1830, #1)