Trans-anethole ameliorates obesity via induction of browning in white adipocytes and activation of brown adipocytes Biochimie (IF 3.112) Pub Date : 2018-05-24 Nam Hyeon Kang, Sulagna Mukherjee, Taesun Min, Sun Chul Kang, Jong Won Yun
Non-stereoselective decomposition of (±)-S-alk(en)yl-L-cysteine sulfoxides to antibacterial thiosulfinates catalyzed by C115H mutant methionine γ-lyase from Citrobacter freundii Biochimie (IF 3.112) Pub Date : 2018-05-25 Vitalia Kulikova, Elena Morozova, Alexei Rodionov, Vasily Koval, Natalya Anufrieva, Svetlana Revtovich, Tatyana Demidkina
S-Alk(en)yl-L-cysteine sulfoxides, initially found in plants of the genus Allium, are converted to antimicrobial thiosulfinates by pyridoxal 5'-phosphate(PLP)-dependent alliinase (EC 126.96.36.199). It was found that methionine γ-lyase (MGL, EC 188.8.131.52) catalyzes the β-elimination reaction of (±)-S-alk(en)yl-L-cysteine sulfoxides to yield thiosulfinates. The efficient catalyst for the production of thiosulfinates, C115H mutant MGL, developed in our previous work, cleaves S-alk(en)yl-L-cysteine sulfoxides more effectively than the wild type enzyme. Thiosulfinates generated by the C115H MGL/sulfoxide system have demonstrated growth inhibition of Gram-positive, Gram-negative bacteria and clinical isolates of pathogenic bacteria from mice. In search of a more effective system for production of antibacterial thiosulfinates we synthesized S-substituted analogues of L-cysteine sulfoxide with a longer side chains – (±)-S-propyl-L-cysteine sulfoxide ((±)-propiin) and (±)-S-n-butyl-L-cysteine sulfoxide ((±)-butiin) and determined catalytic parameters of the β-elimination reaction of two sulfoxides. It was found that C115H MGL cleaves (±)-propiin with the highest rate, as compared to other (±)-S-alk(en)yl-L-cysteine sulfoxides. Studies on interaction of the enzyme with (+)- or (-)-S-alk(en)yl-L-cysteine sulfoxides revealed that C115H MGL can decompose both diastereomers equally. The antibacterial activity of the mixture of the mutant MGL with (±)-propiin is comparable with those of the mixtures with S-allyl-L-cysteine sulfoxide (alliin) and S-methyl-L-cysteine sulfoxide (methiin). The results make MGL/sulfoxide system more advantageous in preparing antibacterial thiosulfinates as compared to alliinase-based system, which preferably cleaves naturally occurring (+)-sulfoxides.
Proteomic analysis of Tn-bearing glycoproteins from different stages of melanoma cells reveals new biomarkers Biochimie (IF 3.112) Pub Date : 2018-05-24 Dorota Hoja-Łukowicz, Sabina Szwed, Piotr Laidler, Anna Lityńska
Chlorogenic acid ameliorates alcohol-induced liver injuries through scavenging reactive oxygen species Biochimie (IF 3.112) Pub Date : 2018-05-19 Hyunjin Kim, Jeong Hoon Pan, Sung Hwan Kim, Jin Hyup Lee, Jeen-Woo Park
Review article A translational view of cells' secretome analysis - from untargeted proteomic to potential circulating biomarkers Biochimie (IF 3.112) Pub Date : 2018-05-18 Sandra I. Anjo, Bruno Manadas
Type I collagen induces mesenchymal cell differentiation into myofibroblasts through YAP-induced TGF-β1 activation Biochimie (IF 3.112) Pub Date : 2018-05-17 Xiaoling Liu, Xinyu Long, Weiwei Liu, Yeli Zhao, Toshihiko Hayashi, Masayuki Yamato, Kazunori Mizuno, Hitomi Fujisaki, Shunji Hattori, Shin-ichi Tashiro, Takaaki Ogura, Yuji Atsuzawa, Takashi Ikejima
In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-β1) promote differentiation into myofibroblast from mesenchymal cells, leading to extracellular matrix (ECM) remodeling or active synthesis, deposition or degradation of ECM components. A major component of ECM, type I collagen (col I) triple helical molecules assemble into fibrils or are denatured to gelatin without triple-helicity in remodeling. However, whether changes of ECM components in remodeling have influence on mesenchymal cell differentiation remains elusive. This study adopted three states of collagen I existing in ECM remodeling: molecular collagen, fibrillar collagen and gelatin to see what are characteristics in the effects on two cell lines of mesenchymal origin, murine 3T3-L1 embryonic fibroblast and murine C2C12 myoblasts. The results showed that all three forms of collagen I were capable of inducing these two cells to differentiate into myofibroblasts characterized by increased expression of alpha-smooth muscle actin (α-SMA) mRNA. The expression of α-SMA is positively regulated by TGF-β1. Nuclear translocation of Yes-associated protein (YAP) is involved in this process. Focal adhesion kinase (FAK) is activated in the cells cultured on molecular collagen-coated plates, contributing to YAP activation. On the other hand, in the cells cultured on fibrillar collagen gel or gelatin-coated plates, oxidative stress but not FAK induce YAP activation. In conclusion, the three physicochemically distinct forms of col I induce the differentiation of mesenchymal cells into myofibroblasts through different pathways.
The amyloidogenicity of a C-terminal region of TDP-43 implicated in Amyotrophic Lateral Sclerosis can be affected by anions, acetylation and homodimerization Biochimie (IF 3.112) Pub Date : 2018-05-08 Archana Prasad, Vishwanath Sivalingam, Vidhya Bharathi, Amandeep Girdhar, Basant K. Patel
Importance of genetic background of oxysterol signaling in cancer Biochimie (IF 3.112) Pub Date : 2018-05-07 P. Holy, A. Kloudova, P. Soucek
Bioinformatics analyses and in vitro evidence for five and six stacked G-quadruplex forming sequences Biochimie (IF 3.112) Pub Date : 2018-05-04 Martin Bartas, Václav Brázda, Václav Karlický, Jiří Červeň, Petr Pečinka
Quadruplexes are noncanonical DNA structures that arise in guanine rich loci and have important biological functions. Classically, quadruplexes contain four stacked intramolecular G-tetrads. Surprisingly, although some algorithms allow searching for longer than 4G tracts for quadruplex formation, these have not yet been systematically studied. Therefore, we analyzed the human genome for sequences that are predicted to adopt stacked intramolecular G-tetrads with greater than four stacks. The data provide evidence for numerous G-quadruplexes that contain five or six stacked intramolecular G-tetrads. These sequences are predominantly found in known gene regulatory regions. Electrophoretic mobility assays and circular dichroism spectroscopy indicate that these sequences form quadruplex structures in vitro under physiological conditions. The localization and in vitro stability of these G-quadruplexes indicate their potentially important roles in gene regulation and their potential for therapeutic applications.
Unusual interaction of human apurinic/apyrimidinic endonuclease 1 (APE1) with abasic sites via the Schiff-base-dependent mechanism Biochimie (IF 3.112) Pub Date : 2018-05-03 Ekaterina S. Ilina, Svetlana N. Khodyreva, Olga I. Lavrik
Clustered apurinic/apyrimidinic (AP) sites are more cytotoxic than isolated AP lesions because double strand breaks (DSB) can be formed during repair of closely positioned bistranded AP sites. Formation of DSB due to simultaneous cleavage of bistranded AP sites may be regulated by proteins specifically interacting with this complex lesion. A set of AP DNA duplexes containing AP sites in both strands in different mutual orientation (BS-AP DNAs) was used for search in the extracts of human cells proteins specifically recognizing clustered AP sites. A protein, which formed the Schiff-base-dependent covalent products having an apparent molecular mass of 50 kDa with the subset of BS-AP DNAs, was identified by mass spectrometry as apurinic/apyrimidinic endonuclease 1 (APE1). The identity of trapped protein was confirmed by Western blot analysis with anti-APE1 antibodies. Purified recombinant human APE1 is also capable of forming the 50 kDa-adducts with efficiency of BS-AP DNAs cross-linking to APE1 being dependent on the mutual orientation of AP sites. In spite of formation of the Schiff-base-dependent intermediate, which is prerequisite for the β-elimination mechanism, APE1 is unable to cleave AP sites. APE1 lacking the first 34 amino acids at the N-terminus, unlike wild type enzyme, is unable to form cross-links with BS-AP DNAs that testifies to the involvement of disordered N-terminal extension, which is enriched in lysine residues, in the interaction with AP sites. The yield of APE1-AP DNA cross-links was found to correlate with the enzyme amount in the extracts estimated by the immunochemical approach; therefore the BS-AP DNA-probes can be useful for comparative analysis of APE1 content in cell extracts.
Sabellastarte magnifica Carboxypetidase Inhibitor: the first Kunitz inhibitor simultaneously interacting with carboxypeptidases and serine proteases Biochimie (IF 3.112) Pub Date : 2018-05-03 Mey Ling Reytor González, Maday Alonso-del-Rivero Antigua, Lizbeth Hedstrom, Petr Kuzmič, José Ricardo Pires
Multi-domain inhibitors capable to block the activity of different classes of proteases are not very common in nature. However, these kinds of molecules are attractive systems for biomedical or biotechnological applications, where two or more different targets need to be neutralized. SmCI, the Sabellastarte magnifica Carboxypeptidase Inhibitor, is a tri-domain BPTI-Kunitz inhibitor capable to inhibit serine proteases and A-like metallocarboxypeptidases. The BPTI-Kunitz family of proteins includes voltage gated channel blockers and inhibitors of serine proteases. SmCI is therefore, the only BPTI-Kunitz protein capable of inhibiting metallocarboxypeptidases. The X-ray structure of the SmCI-carboxypeptidase A complex previously obtained by us, revealed that this enzyme interacts with SmCI N-tail. In the complex, the reactive loops for serine protease inhibition remain fully exposed to the solvent in each domain, suggesting SmCI can simultaneously interact with multiple serine proteases. The twofold goals of this study were: i) to establish serine proteases-SmCI binding stoichiometry, given that the inhibitor is comprised of three potential binding domains; and ii) to determine whether or not SmCI can simultaneously bind both classes of enzymes, to which it binds individually. Our experimental approach included a variety of techniques for the study of protein-protein interactions, using as model enzymes pancreatic trypsin, elastase and carboxypeptidase A. In particular, we combined information obtained from gel filtration chromatography, denaturing electrophoresis, nuclear magnetic resonance spectroscopy and enzyme inhibition assays. Our results show that SmCI is able to bind three trypsin molecules under saturating conditions, but only one elastase interacts with the inhibitor. Additionally, we demonstrated that SmCI can bind serine proteases and carboxypeptidases at the same time (at least in the ratio 1:1:1), becoming the first protease inhibitor that simultaneously block these two mechanistic classes of enzymes.
Biological and regulatory roles of acid-induced small RNA RyeC in Salmonella Typhimurium Biochimie (IF 3.112) Pub Date : 2018-05-03 Daniel Ryan, Mohana Mukherjee, Ritu Nayak, Ria Dutta, Mrutyunjay Suar
Salmonella Typhimurium is an enteric pathogen that has evolved masterful strategies to enable survival under stress conditions both within and outside a host. The acid tolerance response (ATR) is one such mechanism that enhances the viability of acid adapted bacteria to lethal pH levels. While numerous studies exist on the protein coding components of this response, there is very little data on the roles of small RNAs (sRNAs). These non-coding RNA molecules have recently been shown to play roles as regulators of bacterial stress response and virulence pathways. They function through complementary base pairing interactions with target mRNAs and affect their translation and/or stability. There are also a few that directly bind to proteins by mimicking their respective targets. Here, we identify several sRNAs expressed during the ATR of S. Typhimurium and characterize one highly induced candidate, RyeC. Further, we identify ptsI as a trans-encoded target that is directly regulated by this sRNA. From a functional perspective, over-expression of RyeC in Salmonella produced a general attenuation of several in vitro phenotypes including acid survival, motility, adhesion and invasion of epithelial cell lines as well as replication within macrophages. Together, this study highlights the diverse roles played by sRNAs in acid tolerance and virulence of S. Typhimurium.
Exploring the biophysical properties of phytosterols in the plasma membrane for novel cancer prevention strategies Biochimie (IF 3.112) Pub Date : 2018-05-03 Omar Fakih, Didem Sanver, David Kane, James L. Thorne
Cancer is a global problem with no sign that incidences are reducing. The great costs associated with curing cancer, through developing novel treatments and applying patented therapies, is an increasing burden to developed and developing nations alike. These financial and societal problems will be alleviated by research efforts into prevention, or treatments that utilise off-patent or repurposed agents. Phytosterols are natural components of the diet found in an array of seeds, nuts and vegetables and have been added to several consumer food products for the management of cardio-vascular disease through their ability to lower LDL-cholesterol levels. In this review, we provide a connected view between the fields of structural biophysics and cellular and molecular biology to evaluate the growing evidence that phytosterols impair oncogenic pathways in a range of cancer types. The current state of understanding of how phytosterols alter the biophysical properties of plasma membrane is described, and the potential for phytosterols to be repurposed from cardio-vascular to oncology therapeutics. Through an overview of the types of biophysical and molecular biology experiments that have been performed to date, this review informs the reader of the molecular and biophysical mechanisms through which phytosterols could have anti-cancer properties via their interactions with the plasma cell membrane. We also outline emerging and under-explored areas such as computational modelling, improved biomimetic membranes and ex vivo tissue evaluation. Focus of future research in these areas should improve understanding, not just of phytosterols in cancer cell biology but also to give insights into the interaction between the plasma membrane and the genome. These fields are increasingly providing meaningful biological and clinical data but iterative experiments between molecular biology assays, biosynthetic membrane studies and computational membrane modelling improve and refine our understanding of the role of different sterol components of the plasma membrane.
Phosphorylation-Induced Conformational Changes of Photoactivated Rhodopsin Probed by Fluorescent Labeling at Cys140 and Cys316 Biochimie (IF 3.112) Pub Date : 2018-05-04 Sheerly Rodríguez, May-Li Silva, Gustavo Benaím, José Bubis
In order to monitor conformational changes following photoactivation and phosphorylation of bovine rhodopsin, the two reactive sulfhydryl groups at Cys140 and Cys316 were specifically labeled with the monobromobimane (mBBr) fluorophore. Although alterations in conformation after light exposure of rhodopsin were not detected by fluorescence excitation scans (300-450 nm) of the mBBr-labeled protein, the fluorescence signal was reduced ∼ 90% in samples containing photoactivated phosphorhodopsin. Predominant labeling at either Cys140 or Cys316 in light-activated and phosphorylated rhodopsin merely generated a decrease of ∼ 38% and 28%, respectively, in the fluorescence excitation intensity. Thus, neither mBBr-modified Cys140 nor mBBr-modified Cys316 were involved single-handedly in the remarkable fall seen on the signal following phosphorylation of the protein; rather, the incorporation of phosphate groups on the mBBr-labeled light-activated rhodopsin appeared to affect its fluorescence signal in a cooperative or synergistic manner. These findings demonstrated that the phosphorylation of specific hydroxyl groups at the carboxyl terminal tail of rhodopsin causes definite conformational changes in the three-dimensional fold of the protein. Apparently, amino acid residues that are buried in the interior of the inactive protein become accessible following bleaching and phosphorylation of rhodopsin, quenching in turn the fluorescence excitation signal of mBBr-modified rhodopsin.
Notch3 is involved in adipogenesis of human adipose-derived stromal/stem cells Biochimie (IF 3.112) Pub Date : 2018-04-27 Demi A. Sandel, Mengcheng Liu, Ngozi Ogbonnaya, Jamie J. Newman
Human adipose-derived stromal/stem cells (hASCs) have tremendous therapeutic potential and the ability to offer insight into human development and disease. Here we subject human ASCs to siRNA-mediated knockdown of Notch3 cultured under both self-renewing and adipogenic differentiation conditions. Self-renewal was monitored by assessing viability and proliferation rates through staining and alamarBlue assays, respectively. Adipogenesis was measured through Oil-Red O staining, western blot and quantitative real-time RT-PCR that determined expression levels of multipotency and adipogenic markers over time. Notch3 was expressed in self-renewing hASCs but knockdown, as validated by qRT-PCR and western blot, showed no impact on cell viability, as measured through live-dead staining, or cell proliferation rates, as measured through alamarBlue assays. However, as Notch3 expression was observed to increase during adipogenesis, in the absence of Notch3 there was a significant increase in hASC adipogenesis as demonstrated through an increased number of lipid vesicles, and increased expression of adipogenic markers ppar-γ, adiponectin, fabp4, and plin2. Although Notch3 is only one of four Notch receptors expressed on the surface of hASCs, this receptor appears important for proper regulation of adipogenic differentiation, possibly serving as a negative regulator to prevent inappropriate adipogenesis or promote other lineage commitments of ASCs.
Increase in liver cytosolic lipases activities and VLDL-TAG secretion rate do not prevent the non-alcoholic fatty liver disease in cafeteria diet-fed rats Biochimie (IF 3.112) Pub Date : 2018-04-26 Anderson Fernandes de Melo, Carolina Campos Lima Moreira, Camila Ferreira Sales, Thiago Rentz, Helena Fonseca Raposo, Maria Antonieta Rissato Garófalo, Leida Maria Botion, Isis do Carmo Kettelhut, Helena Coutinho Franco de Oliveira, Valéria Ernestânia Chaves
We have previously shown that the cafeteria diet increases body fat mass, triacylglycerol (TAG) and insulin plasma levels, glucose uptake by white and brown adipose tissues, as well as the sympathetic activity to both adipose tissues in Wistar rats. The metabolic pathways responsible for the development of non-alcoholic fatty liver disease (NAFLD) were examined in cafeteria diet-fed rats. After 3 weeks offering cafeteria diet, we evaluated: (i) activity of the sympathetic nervous system by norepinephrine turnover rates; (ii) de novo fatty acid synthesis in vivo using 3H2O; (iii) secretion of very low density lipoprotein (VLDL)-TAG secretion measuring serum TAG levels after administration of lipase lipoprotein inhibitor, (iv) liver cytosolic lipases activity and (v) liver mRNA expression of enzymes involved in lipids secretion and oxidation by RT-PCR. The cafeteria diet induced an increase in TAG (120%) and cholesterol (30%) liver contents. Cafeteria diet did not change the sympathetic nervous system activity to liver, but induced a marked increase in the lipogenesis (approximately four-fold) and significant increase in cytosolic lipases activities (46%) and VLDL-TAG secretion (22%) compared to control diet-fed rats. The cafeteria diet also increased the microsomal triglyceride transfer protein (30%) and carnitine palmitoyltransferase I (130%) mRNA expression but decreased the apolipoprotein B100 (26%) mRNA expression. Our findings demonstrate that the increase in the cytosolic lipases activities and VLDL-TAG secretion rates were not able to compensate for the increased lipogenesis rates induced by the cafeteria diet, resulting in NAFLD.
Ophiopogonin D improves osteointegration of titanium alloy implants under diabetic conditions by inhibition of ROS overproduction via Wnt/β-catenin signaling pathway Biochimie (IF 3.112) Pub Date : 2018-04-26 Xiang-Yu Ma, Xin-Xin Wen, Xiao-Jiang Yang, Da-Peng Zhou, Qiong Wu, Ya-Fei Feng, Hai-Jiao Ding, Wei Lei, Hai-Long Yu, Bing Liu, Liang-Bi Xiang, Tian-Sheng Wang
A high failure rate of titanium implants in diabetic patients has been indicated in clinical evidences. Excessive oxidative stress at the bone-implant interface plays an important role in the impaired osteointegration under diabetic conditions. While the underlying mechanisms remain unknown and the targeted treatments are urgently needed. Ophiopogonin D (OP-D), isolated from Chinese herbal Radix Ophiopogon japonicus, is generally reported to be a potent antioxidant agent. In the present study, we hypothesized that OP-D exerted promotive effects on osteointegration against oxidative stress, and investigated the underlying mechanisms associated with alteration of Wnt/β-catenin signaling pathway. Rabbit osteoblasts incubated on titanium alloy implant were co-cultured with normal serum (NS), diabetic serum (DS), DS + OP-D, DS + NAC (a potent ROS inhibitor) and DS + OP-D + Dkk1 (a Wnt inhibitor) for examinations of osteoblast behaviors. For in vivo study, titanium alloy implants were implanted into the femoral condyle defects on diabetic rabbits. Results demonstrated that diabetes-induced oxidative stress resulted in osteoblast dysfunctions and apoptotic injury at the bone-implant interface, concomitant with the inactivation of Wnt/β-catenin signaling. Importantly, OP-D administration attenuated oxidative stress, directly reactivating Wnt/β-catenin signaling. Osteoblast dysfunctions were thus reversed as evidenced by improved osteoblast adhesion, proliferation and differentiation, and ameliorated apoptotic injury, exerting similar effects to NAC treatment. In addition, the positive effects afforded by OP-D were confirmed by improved osteointegration and oetogenesis within the titanium alloy implants in vivo by Micro-CT and histological analyses. Furthermore, the pro-osteogenic effects of OP-D were almost completely abolished by the Wnt inhibitor Dkk1. These results demonstrated, for the first time, OP-D administration alleviated the damaged osteointegration of titanium alloy implants under diabetic conditions by means of inhibiting oxidative stress via a Wnt/β-catenin-dependent mechanism. The OP-D administration would become a reliable treatment strategy for implant failure therapy in diabetics due to the optimal anti-oxidative and pro-osteogenic properties.
In vitro oxidized HDL and HDL from type 2 diabetes patients have reduced ability to efflux oxysterols from THP-1 macrophages Biochimie (IF 3.112) Pub Date : 2018-04-26 Yinan Chena, Maud Arnal-Levron, Françoise Hullin-Matsuda, Carole Knibbe, Philippe Moulin, Céline Luquain-Costaz, Isabelle Delton
Oxidized LDL (OxLDL) that are enriched in products of lipid peroxidation including oxysterols have been shown to induce cellular oxidative stress and cytotoxicity therefore accelerating atheroma plaque formation. Upon oxLDL exposure of THP-1 macrophages, intracellular oxidation of LDL derived-cholesterol as well as endogenous cholesterol was increased. The oxysterols intracellularly produced were efficiently exported to HDL whereas apolipoprotein A1 was inefficient. These findings prompted us to investigate the consequences of modification of HDL by oxidation and glycation as observed in type 2 diabetes with respect to oxysterol and cholesterol efflux. We show that efflux of oxysterols was significantly impaired after in vitro oxidation and glycoxidation of HDL whereas glycation alone had no impact. Cholesterol efflux was only slightly decreased by oxHDL or glycoxidized HDL and not changed with glycated HDL. The defect of HDL towards oxysterol efflux was also observed with HDL isolated from diabetic subjects as compared to healthy controls. These findings support a deleterious cellular retention of oxysterols due to dysfunctional HDL in type 2 diabetes.
Determination of the binding properties of p-cresyl glucuronide to human serum albumin Biochimie (IF 3.112) Pub Date : 2018-04-26 Dan Yi, Elisa Bernardes Monteiro, Stéphane Chambert, Hédi A. Soula, Julio B. Daleprane, Christophe O. Soulage
p-Cresyl glucuronide (p-CG) is a by-product of tyrosine metabolism that accumulates in patients with end-stage renal disease. p-CG binding to human serum albumin in physiological conditions (37°C, pH 7.40) was studied by ultrafiltration (MWCO 10 kDa) and data were analyzed assuming one binding site. The estimated value of the association constant was 2.77×103 M-1 and a maximal stoichiometry of 3.80 mol per mole. At a concentration relevant for end-stage renal patients, p-CG was 23% bound to albumin. Competition experiments, using fluorescent probes, demonstrated that p-CG did not bind to Sudlow's site I or site II. The p-CG did not interfere with the binding of p-cresyl-sulfate or indoxyl sulfate to serum albumin.
Cellular mechanism of resistance of human colorectal adenocarcinoma cells against apoptosis-induction by Russell's Viper venom L-amino acid oxidase (Rusvinoxidase) Biochimie (IF 3.112) Pub Date : 2018-04-25 Ashis K. Mukherjee, Anthony J. Saviola, Stephen P. Mackessy
The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-205) cells against apoptosis induction by Rusvinoxidase, an L-amino acid oxidase purified from Russell's Viper venom (RVV). The significantly lower cytotoxicity as well as apoptotic activity of Rusvinoxidase towards Colo-205 cells (compared to MCF-7 breast cancer cells) is correlated with lower depletion of cellular glutathione content and increased down-regulation of catalase activity of Colo-205 cells following Rusvinoxidase treatment. Exposure to Rusvinoxidase subsequently diminished reactive oxygen species (ROS) production and failed to impair mitochondrial membrane potential, resulting in apoptosis induction resistance in Colo-205 cells. Further, higher expression levels of caspase 8, compared to caspase 9, indicate that Rusvinoxidase preferentially triggers the extrinsic pathway of apoptosis in Colo-205 cells. A time-dependent lower ratio of the relative expression of Bax and Bcl-xL (pro- and anti-apoptotic proteins) in Colo-205 cells, compared to our previous study on MCF-7 cells, unambiguously supports a higher cellular resistance mechanism in Colo-205 cells against Rusvinoxidase-induced apoptosis.
MiR-98 suppresses the effects of tumor-associated macrophages on promoting migration and invasion of hepatocellular carcinoma cells by regulating IL-10 ☆ Biochimie (IF 3.112) Pub Date : 2018-04-24 Li Lei, Sun Pengfei, Zhang Chengsheng, Li Zongchao, Zhou Wuyuan
We aim to investigate the role of miR-98-mediated macrophage polarization in hepatocellular carcinoma (HCC) progression and to explore the underlying mechanism. A total of 25 paired HCC and matched adjacent normal tissues (ANTs) were collected. We incubated human blood monocytes isolated from healthy male donors with culture medium collected from HepG2 cells for 7 days. The mRNA and protein expression was detected by qRT-PCR and Western blot, respectively. Levels of cytokines secreted in culture medium were measured using the specific ELISA kits. The miR-98 mimic/inhibitor was transfected to explore the role of miR-98 in HCC-conditioned tumor-associated macrophages (TAMs). HepG2 cells were then cultured with condition medium from HCC-conditioned TAMs pretreated with miR-98 mimic/inhibitor, and cell migration and invasion assays were performed. Luciferase reporter assay was performed to analyze the interaction between miR-98 and interleukin (IL)-10. Our results showed that miR-98 was downregulated and IL-10 was upregulated in HCC tissues and HCC-conditioned TAMs. Further studies identified that IL-10 was a direct target gene of miR-98 in HCC-conditioned TAMs. Moreover, miR-98 regulated the levels of inflammatory cytokines in HCC-conditioned TAMs. HCC-conditioned TAMs pretreated with miR-98 regulated migration and invasion of HepG2 cells in vitro, and the effects were significantly reversed by IL-10. In conclusion, miR-98 not only regulated expression of inflammatory cytokines in HCC-conditioned TAMs, but also modulated the capacity of HCC-conditioned TAMs to regulate HepG2 cell migration and invasion, at least in part, by targeting IL-10. As a result, miR-98 may suppress the progress of HCC.
The pericyte secretome: Potential impact on regeneration Biochimie (IF 3.112) Pub Date : 2018-04-23 Abderahim Gaceb, Marco Barbariga, Ilknur Özen, Gesine Paul
Personalized and regenerative medicine is an emerging therapeutic strategy that is based on cell biology and biomedical engineering used to develop biological substitutes to maintain normal function or restore damaged tissues and organs. The secretory capacities of different cell types are now explored as such possible therapeutic regenerative agents in a variety of diseases. A secretome can comprise chemokines, cytokines, growth factors, but also extracellular matrix components, microvesicles and exosomes as well as genetic material and may differ depending on the tissue and the stimulus applied to the cell. With regard to clinical applications, the secretome of mesenchymal stem cells (MSC) is currently the most widely explored. However, other cell types such as pericytes may have similar properties as MSC and the potential therapeutic possibilities of these cells are only just beginning to emerge. In this review, we will summarize the currently available data describing the secretome of pericytes and its potential implications for tissue regeneration, whereby we especially focus on brain pericytes as potential new target cell for neuroregeneration and brain repair.
EBI2 – Sensor for Dihydroxycholesterol Gradients in Neuroinflammation Biochimie (IF 3.112) Pub Date : 2018-04-22 Florian Kurschus, Florian Wanke
Dihydroxycholesterols such as 7α,25-dihydroxysterols (7α,25-OHC) and 7α,27-OHC are generated from cholesterol by the enzymes CH25H, CYP7B1 and CYP27A1 in steady state but also in the context of inflammation. The G-protein coupled receptor (GPCR) Epstein-Barr virus-induced gene 2 (EBI2), also known as GPR183, senses these oxysterols and induces chemotactic migration of immune cells towards higher concentrations of these ligands. We recently showed that these ligands are upregulated in the CNS in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis and that EBI2 enhanced early infiltration of encephalitogenic T cells into the CNS. In this short-review we discuss the role of dihydroxysterol-sensing by immune cells in neuroinflammation.
Functional characterization and molecular modeling of the mutations in CYP21A2 gene from patients with Congenital Adrenal Hyperplasia Biochimie (IF 3.112) Pub Date : 2018-04-22 Ragini Khajuria, Rama Walia, Anil Bhansali, Rajendra Prasad
Steroid 21-Hydroxylase deficiency is an inherited autosomal recessive metabolic disorder of the adrenal steroidogenesis caused due to mutations in the CYP21A2 gene in 95% of CAH cases. Notably, the de novo mutations arise at the rate of 3-5%, therefore the functional characterization is of utmost importance for categorization of the novel mutations. Herein, we have functionally characterized the CYP21A2 missense mutations viz., p. F306V and p. H365 N. Notably, both the mutations were harbored by the patients exhibiting the non classical phenotype. We followed the approach of in vitro characterization of the mutant proteins expressed in COS7 cells. Of note, all the mutant constructs exhibited reduced residual enzyme activity fraternized with altered kinetic constants accompanied by higher requirement for the activation energy. Further, there was reduced protein expression in the mutant constructs to that of the wild-type. Molecular modeling suggested alteration in the structure-function relationship of the protein due to mutations. The evidence suggested by the in vitro and the in silico characterization of mutations directed us to conclude that both, p. F306V and p. H365 N should be considered as non classical CAH causing mutations. Conceivably, the knowledge about the functional consequences of the mutations is the basis for improved genetic counseling with respect to prognosis and therapeutic implications.
The effect of oxysterols on nerve impulses Biochimie (IF 3.112) Pub Date : 2018-04-21 Maryem Bezine, Amira Namsi, Randa Sghaier, Rym Ben Khalifa, Haithem Hamdouni, Fatiha Brahmi, Iham Badreddine, Wafa Mihoubi, Thomas Nury, Anne Vejux, Amira Zarrouk, Jérôme de Sèze, Thibault Moreau, Boubker Nasser, Gérard Lizard
The propagation of nerve impulses in myelinated nerve fibers depends on a number of factors involving the myelin and neural axons. In several neurodegenerative diseases, nerve impulses can be affected by the structural and biochemical characteristics of the myelin sheath and the activity of ion channels located in the nodes of Ranvier. Though it is generally accepted that lipid disorders are involved in the development of neurodegenerative diseases, little is known about their impact on nerve impulses. Cholesterol oxide derivatives (also called oxysterols), which are either formed enzymatically or as a result of cholesterol auto-oxidation or both, are often found in abnormal levels in the brain and body fluids of patients with neurodegenerative diseases. This leads to the question of whether these molecules, which can accumulate in the plasma membrane and influence its structure and functions (fluidity, membrane proteins activities, signaling pathways), can have an impact on nerve impulses. It is currently thought that the ability of oxysterols to modulate nerve impulses could be explained by their influence on the characteristics and production of myelin as well as the functionality of Na+ and K+ channels.
Exposing mesenchymal stem cells to chondroitin sulphated proteoglycans reduces their angiogenic and neuro-adhesive paracrine activity Biochimie (IF 3.112) Pub Date : 2018-04-19 Chelsea R. Wood, Ibtesam R.T. Al Delfi, John F. Innes, Peter Myint, William E.B. Johnson
The multifactorial complexity of spinal cord injuries includes the formation of a glial scar, of which chondroitin sulphated proteoglycans (CSPG) are an integral component. Previous studies have shown CSPG to have inhibitory effects on endothelial and neuronal cell growth, highlighting the difficulty of spinal cord regeneration. Mesenchymal stem/stromal cells (MSC) are widely used a cell therapy, and there is mounting evidence for their angiogenic and neurotrophic paracrine properties. However, in vivo studies have observed poor engraftment and survival of MSC when injected into SCI. Currently, it is not known whether increasing CSPG concentrations seen after SCI may affect MSC; therefore we have investigated the effects of CSPG exposure to MSC in vitro. CSPG-mediated inhibition of MSC adhesion was observed when MSC where cultured on substrates of increasing CSPG concentration, however MSC viability was not affected even up to five days of culture. Culture conditioned medium harvested from these cultures (primed MSC CM) was used as both culture substrata and soluble medium for EA. hy926 endothelial cells and SHSY5Y neuronal cells. MSC CM was angiogenic, promoting endothelial cell adhesion, proliferation and tubule formation. However, exposing MSC to CSPG reduced the effects of CSPG-primed MSC CM on endothelial cell adhesion and proliferation, but did not reduce MSC-induced endothelial tubule formation. Primed MSC CM also promoted neuronal cell adhesion, which was reduced following exposure to CSPG. There were no marked differences in neurite outgrowth in MSC CM from CSPG primed MSC cultures versus control conditions, although non-primed MSC CM from the same donors was found to significantly enhance neurite outgrowth. Taken together, these studies demonstrate that MSC are resilient to CSPG exposure, but that there is a marked effect of CSPG on their paracrine regenerative activity. The findings increase our understanding of how the wound microenvironment after SCI can mitigate the beneficial effects of MSC transplantation.
Chemistry, biochemistry, metabolic fate and mechanism of action of 6-oxo-cholestan-3β,5α-diol (OCDO), a tumor promoter and cholesterol metabolite Biochimie (IF 3.112) Pub Date : 2018-04-12 Marc Poirot, Regis Soules, Arnaud Mallinger, Florence Dalenc, Sandrine Silvente-Poirot
Oxygenation products of cholesterol, named oxysterols, were suspected since the 20th century to be involved in carcinogenesis. Among the family of oxysterol molecules, cholesterol-5,6-epoxides (5,6-EC) retained the attention of scientists because they contain a putative alkylating epoxide group. However, studies failed into demonstrating that 5,6-EC were direct carcinogens and revealed a surprising chemical stability and unreactivity towards nucleophiles in standard conditions. Analyses of 5,6-EC metabolism in normal cells showed that they were extensively transformed into cholestane-3β,5α,6β-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH). Studies performed in cancer cells showed that CT was additionally metabolized into an oxysterol identified as the 6-oxo-cholestan-3β,5α-diol (OCDO), by the 11β-hydroxysteroid dehydrogenase of type 2 (HSD2), the enzyme which inactivates cortisol into cortisone. Importantly, OCDO was shown to display tumor promoter properties in breast cancers, by binding to the glucocorticoid receptor, and independently of their estrogen receptor status, revealing the existence of a new tumorigenic pathway centered on 5,6-EC. In breast tumors from patients, OCDO production as well as the expression of the enzymes involved in the pathway producing OCDO, namely ChEH subunits and HSD2, were higher compared to normal tissues, and overexpression of these enzymes correlate with a higher risk of patient death, indicating that this onco-metabolism is of major importance to breast cancer pathology. Herein, we will review the actual knowledge and the future trends in OCDO chemistry, biochemistry, metabolism and mechanism of action and will discuss the impact of OCDO discovery on new anticancer therapeutic strategies.
EFFECTS OF THE BETA2 AGONIST FORMOTEROL ON ATROPHY SIGNALING, AUTOPHAGY, AND MUSCLE PHENOTYPE IN RESPIRATORY AND LIMB MUSCLES OF RATS WITH CANCER-INDUCED CACHEXIA Biochimie (IF 3.112) Pub Date : 2018-04-12 Anna Salazar-Degracia, Sílvia Busquets, Josep M. Argilés, Núria Bargalló-Gispert, Francisco J. López-Soriano, Esther Barreiro
Dual mechanism of Type VII collagen transfer by bone marrow mesenchymal stem cell extracellular vesicles to recessive dystrophic epidermolysis bullosa fibroblasts Biochimie (IF 3.112) Pub Date : 2018-04-10 Jeffrey McBride, Luis Rodriguez-Menocal, Ambar Candanedo, Wellington Guzman, Marta Garcia-Contreras, Evangelos Badiavas
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe blistering disease resulting from a lack of type VII collagen production. Recent clinical trials have shown efficacy of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of epidermolysis bullosa, including improved basement membrane restructuring and cutaneous wound healing. The mechanism as to how type VII collagen is transferred from donor stem cell to recipient RDEB cells has not been defined. Here, we submit the model that BM-MSC-derived extracellular vesicles serve at least two roles: 1) to help transport type VII collagen within the extracellular space; and 2) to feed RDEB fibroblasts with messenger RNA that codes for type VII collagen, resulting in COL7A1 translation and synthesis of type VII collagen alpha chain proteins by RDEB fibroblasts. Utilizing a chemoselective ligation detection method, we found RDEB cells that were treated simultaneously with BM-MSC EVs and an L-methionine analog, L-homopropargylglycine (HPG), synthesized collagen VII alpha chain protein that contained the alkyne group of HPG to react (i.e. undergo the Click-iT® reaction) with azide-modified Alexa 594, suggesting de novo synthesis of type VII collagen by RDEB fibroblasts. Thus, our results support a model in which BM-MSC EVs help increase type VII collagen levels available to recipient cells by 1) donating BM-MSC type VII collagen protein and 2) inducing RDEB fibroblasts to make their own type VII collagen protein. These findings allow us to hypothesize that the secretome of BM-MSCs could have therapeutic value in the treatment of RDEB-related skin disorders.
EFFECT OF Ca2+ ON THE REDOX POTENTIAL OF HEME a IN CYTOCHROME c OXIDASE Biochimie (IF 3.112) Pub Date : 2018-04-07 Tatiana V. Vygodina, Olga P. Kaminskaya, Alexander A. Konstantinov, Vasily V. Ptushenko
Subunit I of cytochrome c oxidase (CcO) from mitochondria and many bacteria contains a cation binding site (CBS) located at the outer positively charged (P-) aqueous phase not far from heme a. Binding of Ca2+ with the CBS in bovine CcO inhibits activity of the enzyme 2-3 -fold [Vygodina, T., Kirichenko, A. & Konstantinov A.A. (2013) Direct Regulation of Cytochrome c Oxidase by Calcium Ions, PLoS One.8 e74436]. Here we show that binding of Ca2+ at CBS of bovine CcO shifts Em of heme a to the positive by 15-20 mV. Na+ ions that bind to the same site and compete with Ca2+ do not affect Em of heme a and also prevent and reverse the effect of Ca2+. No effect of Ca2+ or EGTA is observed on Em of heme a with the wild type bacterial oxidases from R.sphaeroides or P.denitrificans that contain tightly-bound calcium at the site. In the D477A mutant CcO from P. denitrificans that binds Ca2+ reversibly like the mitochondrial CcO, calcium shifts redox titration curve of heme a to the positive by ∼35-50 mV that is in good agreement with the results of electrostatic calculations; however, as shown earlier, it does not inhibit CcO activity of the mutant enzyme. Therefore the data do not support the proposal that the inhibitory effect of Ca2+ on CcO activity may be explained by the Ca2+-induced shift of Em of heme a. Rather, Ca2+ retards electron transfer by inhibition of charge dislocation in the exit part of the proton channel H in mammalian CcO, that is absent in the bacterial oxidases.
Unique nuclear localization of Nile tilapia (Oreochromis niloticus) Neu4 sialidase is regulated by nuclear transport receptor importin α/β Biochimie (IF 3.112) Pub Date : 2018-04-07 Akinobu Honda, Petros Kingstone Chigwechokha, Yuko Kamada-Futagami, Masaharu Komatsu, Kazuhiro Shiozaki
Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin β and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin.
Chikungunya virus inhibition by peptidomimetic inhibitors targeting virus-specific cysteine protease Biochimie (IF 3.112) Pub Date : 2018-04-07 Harvijay Singh, Rajat Mudgal, Manju Narwal, Ramanjit Kaur, Vedita Anand Singh, Anjali Malik, Madhulika Chaudhary, Shailly Tomar
Chikungunya virus (CHIKV), a mosquito-borne pathogenic virus that reemerged and caused epidemic in the Indian Ocean island of La Réunion, is a potential public health threat. Currently there is no antiviral drug or vaccine commercially available for the treatment of chikungunya fever, which necessitates the urge for an effective antiviral therapy for chikungunya treatment. In the present study, a FRET based protease assay was used to analyze the proteolytic activity of chikungunya nsP2 protease (CHIKV nsP2pro) - an essential viral enzyme, with fluorogenic substrate peptide. This protease assay was used to assess the inhibitory activity of Pep-I (MMsINC® database ID MMs03131094) and Pep-II (MMsINC® database ID MMs03927237), peptidomimetic compounds identified in a previous study by our group. Both compounds inhibited CHIKV nsP2pro with half maximal inhibition concentration (IC50) values of ∼34 μM and ∼42 μM, respectively. Kinetic studies showed that the inhibition constant (Ki) value is 33.34±2.53 μM for Pep-I and 45.89±4.38 μM for Pep-II. Additionally, these two compounds significantly inhibited CHIKV replication in BHK-21 cells at concentrations much lower than their cytotoxic concentrations. Intriguingly, these compounds did not show inhibitory effect on Sindbis virus. This suggests that Pep-I and Pep-II compounds identified as CHIKV nsP2 substrate peptidomimetics, specifically inhibit CHIKV replication.
Reply to Caetano-Anollés et al. comment on “Empirical genome evolution models root the tree of life” Biochimie (IF 3.112) Pub Date : 2018-04-06 Ajith Harish, Charles Kurland
We recently analyzed the robustness of competing evolution models developed to identify the root of the Tree of Life: 1) An empirical Sankoff parsimony (ESP) model (Harish and Kurland, 2017), which is a nonstationary and directional evolution model; and 2) An a priori ancestor (APA) model (Kim and Caetano-Anollés, 2011) that is a stationary and reversible evolution model. Both Bayesian model selection tests as well as maximum parsimony analyses demonstrate that the ESP model is, overwhelmingly, the better model. Moreover, we showed that the APA model is not only sensitive to artifacts, but also that the underlying assumptions are neither empirically grounded nor biologically realistic.
Interaction of fluorescently labeled pyrrole-imidazole polyamide probes with fixed and living murine and human cells Biochimie (IF 3.112) Pub Date : 2018-04-05 Karine Nozeret, François Loll, Gildas Mouta Cardoso, Christophe Escudé, Alexandre S. Boutorine
Pericentromeric heterochromatin plays important roles in controlling gene expression and cellular differentiation. Fluorescent pyrrole-imidazole polyamides targeting murine pericentromeric DNA (major satellites) can be used for the visualization of pericentromeric heterochromatin foci in live mouse cells. New derivatives targeting human repeated DNA sequences (α-satellites) were synthesized and their interaction with target DNA was characterized. The possibility to use major satellite and α -satellite binding polyamides as tools for staining pericentromeric heterochromatin was further investigated in fixed and living mouse and human cells. The staining that was previously observed using the mouse model was further characterized and optimized, but remained limited regarding the fluorophores that can be used. The promising results regarding the staining in the mouse model could not be extended to the human model. Experiments performed in human cells showed chromosomal DNA staining without selectivity. Factors limiting the use of fluorescent polyamides, in particular probe aggregation in the cytoplasm, were investigated. Results are discussed with regards to structure and affinity of probes, density of target sites and chromatin accessibility in both models.
The revelation of selective sphingolipid pathway inhibition mechanism on fumonisin toxin binding to ceramide synthases in susceptible organisms and survival mechanism in resistant species Biochimie (IF 3.112) Pub Date : 2018-04-03 Shikha Sharma, Mushtaq Ahmed, Yusuf Akhter
Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase Biochimie (IF 3.112) Pub Date : 2018-04-03 Marina V. Serebryakova, Yulia V. Bertsova, Svyatoslav S. Sokolov, Alexander A. Kolesnikov, Alexander A. Baykov, Alexander V. Bogachev
One of the three domains of kinetoplastid NADH:fumarate oxidoreductase (FRD) is homologous to bacterial flavin transferase that catalyzes transfer of FMN residue from FAD to threonine in flavoproteins. Leptomonas pyrrhocoris FRD produced in yeast cells, which lack flavin transferase gene in their proteome, reduces fumarate in the presence of NADH and contains an FMN residue covalently linked to a Ser9 residue. The conserved flavinylation motif of FRD, D3(g/s)x(s/t)(s/g)AS9, is similar to the Dxx(s/t)gAT motif recognized by flavin transferase in prokaryotic proteins. Ser9 replacement abolished the flavinylation and fumarate reductase activity of FRD. These findings suggest that the flavinylation is important for the activity of FRD and that this post-translational modification is carried out by the own flavin transferase domain.
Biophysical and in silico interaction studies of aporphine alkaloids with Malonyl-CoA: ACP transacylase (FabD) from drug resistant Moraxella catarrhalis Biochimie (IF 3.112) Pub Date : 2018-03-29 Vijay Kumar, Anchal Sharma, Shivendra Pratap, Pravindra Kumar
Malonyl-CoA:acyl carrier protein transacylase (FabD), being an essential enzyme of the FAS II pathway, is an attractive target for developing broad-spectrum antibiotics. It performs initiation reaction to form malonyl-ACP, which is a key building block in fatty acid biosynthesis. In this study, we have characterized the FabD from drug-resistant pathogen Moraxella catarrhalis (McFabD). More importantly, we have shown the binding of McFabD with three new compounds from the class of aporphine alkaloids. ITC based binding studies have shown that apomorphine is binding to McFabD with a stronger affinity (KD = 4.87 μ&Mgr) as compared to boldine (KD = 7.19 μ&Mgr) and magnoflorine (KD = 11.7 μ&Mgr). The possible mechanism of fluorescence quenching is found to be static with Kq values higher than 1010, which was associated with the ground state complex formation of aporphine alkaloids with McFabD. Conformational changes observed in the secondary and tertiary structure marked by the loss of helical content during the course of interactions. Molecular docking based studies have predicted the binding mode of aporphine alkaloids and it is found that these compounds are interacting in a similar fashion as known inhibitor corytuberine is interacting with McFabD. The analysis of docking poses have revealed that His 210, Leu102, Gln19, Ser101 and Arg 126 are critical residues which may play important role in binding. The growth inhibition assay has shown that apomorphine has better MIC value (4–8 μg/ml) against Moraxella catarrhalis as compared to boldine and magnoflorine. Therefore, the current study suggests that aporphine alkaloids can act as antibacterial agents and possible target of these compounds could be FabD enzyme from the FAS II pathway, and apomorphine scaffold will be more suitable among these compounds for potential development of antibacterial agents.
Overexpression of Egr2 and Egr4 protects rat brains against ischemic stroke by downregulating JNK signaling pathway Biochimie (IF 3.112) Pub Date : 2018-03-24 Rui-Na Niu, Xiao-Ping Shang, Jun-Fang Teng
Objective The purpose of this study was to investigate the effect of Egr2 and Egr4 upregulation on ischemic stroke recovery of rats. Methods In this study, Sprague Dawley (SD) rats assigned at random into control, sham and MCAO (middle cerebral artery occlusion) group were treated accordingly to build MCAO models. The neurological severity scores (NSS) test was applied to assess rats' behavior. Triphenyltetrazolium chloride (TTC) staining reflected infarct areas while Nissl staining revealed the number of neurons. Levels of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6 and tumor necrosis factor [TNF]-α) were judged by enzyme-linked immunosorbent assay (ELISA) in brain and serum tissues. We applied western blot to check the expression of Egr2, Egr4 and JNK/c-JUN (c-Jun N-terminal kinase) pathway. Further grouping of rats were based on various transfection, requiring control, sham, MCAO, MCAO + Egr2 cDNA (complementary DNA), MCAO + Egr4 cDNA, MCAO + Egr2 cDNA + Egr4 cDNA group to observe difference in MCAO recovery and JNK/c-JUN-pathway-related protein expression. Results Under successful modeling of MCAO, western blot results suggested down-regulation of Egr2 and Egr4 and overexpression of pro-inflammatory cytokines. The JNK/c-JUN pathway was activated. On upregulation of Egr2 and Egr4 in infarct areas, neurological function of SD rats recovered along with repressed JNK/c-JUN pathway activation and increased neuron number. Conclusion Upregulation of Egr2 and Egr4 could demote the activation of JNK/c-JUN pathway and the expression of pro-inflammatory cytokines in MCAO rats, so that Egr2 and Egr4 might be potential targets for ischemic stroke in future.
Corrigendum to “DNA Triple Helices: Biological consequences and therapeutic potential” [Biochimie 90/8 (2008) 1117–1130] Biochimie (IF 3.112) Pub Date : 2018-03-23 Aklank Jain, Guliang Wang, Karen M. Vasquez
Lipid transfer protein isolated from noni seeds displays antibacterial activity in vitro and improves survival in lethal sepsis induced by CLP in mice Biochimie (IF 3.112) Pub Date : 2018-03-22 Adson A. Souza, Andrea S. Costa, Dyély C.O. Campos, Andressa H.M. Batista, Gleilton W.P. Sales, Nádia A.P. Nogueira, Keila M.M. Alves, Andrelina Noronha Coelho-de-Souza, Hermógenes D. Oliveira
In the present study, we aimed to evaluate the antibacterial activity of a lipid transfer protein isolated from Morinda citrifolia L. seeds, named McLTP1, and to investigate its effect in the cecal ligation and puncture (CLP) mouse sepsis model. Antimicrobial assays revealed that McLTP1 (12.5–800 μg/mL) significantly reduced Staphylococcus aureus (ATCC 6538P and ATCC 14458) and Staphylococcus epidermidis (ATCC 12228) planktonic growth, reaching maximal inhibition of approximately 50% and 98%, respectively. Furthermore, McLTP1 inhibited biofilm formation of both S. aureus strains, achieving percentages ranging from 39.1% to 69.1% (200–800 μg/mL) for ATCC 6538P and 34.4%–63% (12.5–800 μg/mL) for ATCC 14458. A synergistic interaction between McLTP1 and oxacillin against S. aureus and S. epidermidis was also observed, as determined by fractional inhibitory concentration indices of 0.18 and 0.38, respectively. McLTP1 showed no significant inhibitory effect against Gram-negative bacteria. In the in vivo experiments, sepsis was lethal to 83% of the animals, 72 h after CLP. In contrast, 100% of the animals treated with McLTP1 (8 mg/kg) before (intraperitoneal injection or oral dose) or after (oral dose) CLP were still alive 3 days later. In addition, oral or intraperitoneal administration of McLTP1 (8 mg/kg) significantly reduced the body weight loss, fever, leukocytosis, organ damage, and the level of inflammatory serum cytokines induced by sepsis. In conclusion, McLTP1 could be exploited for its antimicrobial properties, and can be considered a potential therapeutic candidate for the management of clinical sepsis.
Competing trends of ROS and RNS-mediated protein modifications during hypoxia as an alternate mechanism of NO benefits Biochimie (IF 3.112) Pub Date : 2018-03-20 Anamika Gangwar, Subhojit Paul, Yasmin Ahmad, Kalpana Bhargava
Hypoxia, especially altitude associated hypoxia is known to cause severe physiological alterations and life-threatening conditions. Impaired redox balance along with oxidative stress, protein carbonylation and instigation of apoptotic events are common sub-cellular events that follow the hypoxic insult. The role of nitric oxide (NO) is very dynamic and versatile in preventing the ill effects of hypoxia vis-a-vis reacting with oxidative species and causing protein nitrosylation. Although several mechanisms of NO-mediated cytoprotection are known during hypoxic insult, limited evidences are available to support the relationship between two downstream events of oxidative stress, protein carbonylation (caused by carbonyl; CO radical) and protein nitrosylation/nitration (caused by NO/peroxynitrite; ONOO radical). In this study, we investigated an entirely new aspect of NO protection in hypoxia involving cross talk between carbonylation and nitrosylation. Using standard NO inhibitor l-NAME and simulated hypoxic conditions in hypoxia-sensitive cell line H9c2, we evaluated the levels of radicals, cell death, mitochondrial membrane potential, levels of protein nitrosylation, protein nitration and carbonylation and glutathione content. The results were then carefully analyzed in light of NO bioavailability. Our study shows that reducing NO during hypoxia caused cell death via increased degree of carbonylation in proteins. This provides a new aspect of NO benefits which furthers opens new possibilities to explore potential mechanisms and effects of cross-talk between nitrosylation, protein nitration and carbonylation, especially through some common antioxidant mediators such as glutathione and thioredoxin.
Lipidome-wide disturbances of human placental JEG-3 cells by the presence of MEHP Biochimie (IF 3.112) Pub Date : 2018-03-19 Julia Petit, Anaïs Wakx, Sophie Gil, Thierry Fournier, Nicolas Auzeil, Patrice Rat, Olivier Laprévote
During pregnancy, exposure to environmental contaminants can lead to adverse effects on fetal growth and development, especially by targeting the placenta. Di(2-ethylhexyl)phthalate (DEHP), the most abundant chemical used in plastic materials, is known to induce toxicity on animals reproductive system and is suspected to give rise to similar effect in humans. Toxicity of DEHP is due to its main metabolite, MEHP, which is also known to disturb lipid synthesis in several organs. Moreover, mono-(2-ethylhexyl)phtalate (MEHP) is a high affinity ligand of the peroxisome proliferator-activated receptor PPARγ which is essential for placental development and lipid metabolism. In order to investigate possible lipid disruptions induced by MEHP, in the JEG-3 human trophoblast cell line, a differential lipidomic analysis was carried out by UPLC-MS on both exposed and control cells. Our results showed that MEHP induced an important change of JEG-3 cells lipidome, especially in glycerolipids and glycerophospholipids, with a marked accumulation of triacylglycerols. For the first time, our results highlighted adverse effects of MEHP on human placental cells lipidome and thus, its potential effect on placental physiology.
Structural and biochemical characterization of a GH3 Β-glucosidase from the probiotic bacteria Bifidobacterium adolescentis Biochimie (IF 3.112) Pub Date : 2018-03-16 Renata N. Florindo, Valquiria P. Souza, Lívia R. Manzine, Cesar M. Camilo, Sandro R. Marana, Igor Polikarpov, Alessandro S. Nascimento
Bifidobacterium is an important genus of probiotic bacteria colonizing the human gut. These bacteria can uptake oligosaccharides for the fermentative metabolism of hexoses and pentoses, producing lactate, acetate as well as short-chain fatty acids and propionate. These end-products are known to have important effects on human health. β-glucosidases (EC 184.108.40.206) are pivotal enzymes for the metabolism and homeostasis of Bifidobacterium, since they hydrolyze small and soluble saccharides, typically producing glucose. Here we describe the cloning, expression, biochemical characterization and the first X-ray structure of a GH3 β-glucosidase from the probiotic bacteria Bifidobacterium adolescentis (BaBgl3). The purified BaBgl3 showed a maximal activity at 45 °C and pH 6.5. Under the optimum conditions, BaBgl3 is highly active on 4-nitrophenyl-β-d-glucopyranoside (pNPG) and, at a lesser degree, on 4-nitrophenyl-β-d-xylopyranoside (pNPX, about 32% of the activity observed for pNPG). The 2.4 Å resolution crystal structure of BaBgl3 revealed a three-domain structure composed of a TIM barrel domain, which together with α/β sandwich domain accommodate the active site and a third C-terminal fibronectin type III (FnIII) domain with unknown function. Modeling of the substrate in the active site indicates that an aspartate interacts with the hydroxyl group of the C6 present in pNPG but absent in pNPX, which explains the substrate preference. Finally, the enzyme is significantly stabilized by glycerol and galactose, resulting in considerable increase in the enzyme activity and its lifetime. The structural and biochemical studies presented here provide a deeper understanding of the molecular mechanisms of complex carbohydrates degradation utilized by probiotic bacteria as well as for the development of new prebiotic oligosaccharides.
RecF, UvrD, RecX and RecN proteins suppress DNA degradation at DNA double-strand breaks in Escherichia coli Biochimie (IF 3.112) Pub Date : 2018-03-16 Isidoro Feliciello, Davor Zahradka, Ksenija Zahradka, Siniša Ivanković, Nikolina Puc, Damir Đermić
Double strand breaks (DSBs) in E. coli chromosome (such as those induced by gamma rays) are repaired by recombination repair, during which a certain amount of DNA gets degraded. We monitored DNA degradation in gamma-irradiated cells to assess processing of DSBs. DNA degradation in irradiated cells is regulated by RecA protein concentration and its affinity of ssDNA binding, as well as by exonucleases that trim 3’-terminated ss tails. Here we determined the effects of proteins that affect formation and stability of RecA nucleofilaments on DNA degradation and cell survival. RecF and UvrD suppressed DNA degradation through RecA protein function and SOS induction, while also improving gamma survival. RecF and UvrD function in one pathway. Acting along with RecF, RecX suppressed DNA degradation and stimulated gamma-survival, which also depends on RecA protein and SOS induction. Furthermore, we determined a role in DNA degradation of several proteins that participate in DSB repair. RecN was required for DNA repair and for degradation suppression, acting on the RecBCD pathway. Furthermore, we show that SSB protein overproduction did not affect DNA degradation. Inactivation of RecG and RuvABC, proteins that catalyze the postsynaptic phase of recombination repair of DSBs, also did not affect DNA degradation, suggesting that once formed, recombination intermediates are not subject to DNA degradation, and that the postsynaptic phase is an irreversible, single-round process, unlike the presynaptic phase, which is mostly repetitive.
Exosomes as emerging players in cancer biology Biochimie (IF 3.112) Pub Date : 2018-03-16 Nuno Couto, Sergio Caja Galán, Joana Maia, Maria Carolina Strano Moraes, Bruno Costa-Silva
Oncologic diseases do not behave as isolated entities. Instead, they are based on complex systemic networks involving cell-cell communication between cancerous and healthy cells of the host, which may either facilitate or prevent cancer progression. In addition to cell-cell contacts, cells communicate through secreted factors in a process modulated by ligand concentration, receptor availability and synergy amongst several signaling circuits. Of these secreted factors, exosomes, 30 to 150 nm membrane vesicles of endocytic origin released by virtually all cells, have emerged as important cell-cell communication players both in physiological and pathological scenarios by being carriers of all the main biomolecules, including lipids, proteins, DNAs, messenger RNAs and microRNA, and performing intercellular transfer of components, locally and systemically. By acting both in tumor and non-tumor cells, such as fibroblasts, leukocytes, endothelial and progenitor cells, tumor- and non-tumor cells-derived exosomes can modulate tumor growth and invasion, tumor-associated angiogenesis, tissue inflammation and the immune system. In this Review, we summarize the main findings of the literature on the roles of exosomes in mediating interactions between tumor and tumor-associated cells. We also discuss how the molecular composition analysis of circulating exosomes in clinical settings has emerged as an attractive non-invasive source of liquid biopsies for early diagnosis, prognosis and follow-up of patients with oncologic diseases.
Autocrine transforming growth factor-β/activin A-Smad signaling induces hepatic progenitor cells undergoing partial epithelial-mesenchymal transition states Biochimie (IF 3.112) Pub Date : 2018-03-13 Yu Wu, Ze-yang Ding, Guan-nan Jin, Yi-xiao Xiong, Bin Yu, Yi-min Sun, Wei Wang, Hui-fang Liang, Bixiang Zhang, Xiao-ping Chen
Nuclear factor erythroid-2-related factor regulates LRWD1 expression and cellular adaptation to oxidative stress in human embryonal carcinoma cells Biochimie (IF 3.112) Pub Date : 2018-03-12 Jui-Hsiang Hung, Shi-Kae Wee, Hany A. Omar, Chia-Hui Su, Hsing-Yi Chen, Pin-Shern Chen, Chien-Chih Chiu, Ming-Syuan Wu, Yen-Ni Teng
Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is implicated in the regulation of signal transduction, transcription, RNA processing and tumor development. However, LRWD1 transcriptional regulation is not fully understood. This study aimed to investigate the relationship between LRWD1 expression and reactive oxygen species (ROS) level in human embryonal carcinoma cell line, NT2/D1 cells, which will help in understanding the transcriptional regulatory role of ROS in cells. Results showed that the exposure of NT2/D1 cells to various concentrations of hydrogen peroxide (H2O2) and the nitric oxide (NO) donor sodium nitroprusside (SNP) caused a significant increase in the mRNA and protein expression of LRWD1. In addition, LRWD1 promoter luciferase reporter assay, and Chromatin Immunoprecipitation assay (CHIP assay) showed that nuclear factor erythroid-2-related factor (Nrf2) was involved in the regulation of LRWD1 expression in response to oxidative stress. The involvement of Nrf2 was confirmed by shRNA-mediated knockdown of Nrf2 in NT2/D1 cells, which caused a significant decrease in LRWD1 expression in response to oxidative stress. Similarly, LRWD1 knockdown resulted in the accumulation of H2O2 and superoxide anion radical (O2-). Blocking ROS production by N-acetyl cysteine (NAC) protected NT2/D1 shLRWD1cells from H2O2-induced cell death. Collectively, oxidative stress increased LRWD1 expression through Nrf2-dependent mechanism, which plays an important role in cellular adaptation to oxidative stress. These results highlight an evidence, on the molecular level, about LRWD1 transcriptional regulation under oxidative stress.
Cymbopogon citratus and Cymbopogon giganteus essential oils have cytotoxic effects on tumor cell cultures. Identification of citral as a new putative anti-proliferative molecule Biochimie (IF 3.112) Pub Date : 2018-03-06 Bagora Bayala, Imaël H.N. Bassole, Salwan Maqdasy, Silvère Baron, Jacques Simpore, Jean-Marc A. Lobaccaro
Arrangements of nucleotides flanking the start codon in the IRES of the hepatitis C virus in the IRES binary complex with the human 40S ribosomal subunit Biochimie (IF 3.112) Pub Date : 2018-03-01 Elena S. Babaylova, Dmitri M. Graifer, Alexey A. Malygin, Galina G. Karpova
Genomic RNAs of hepatitis C virus (HCV) has an internal ribosome entry site (IRES), a specific highly structured fragment responsible for its non-canonical translation initiation. The HCV IRES contains a major part of the 5′-untranslated region of the viral RNA and a small portion of the open reading frame (ORF). At the first step of initiation, IRES directly binds to 40S ribosomal subunits so that the AUG start codon appears at the P site region without scanning and without involving initiation factors. However, it is still not entirely clear whether the IRES ORF is correctly loaded into the 40S ribosomal mRNA binding channel in the resulting binary complex. To address this issue, we applied site-directed cross-linking using HCV IRES derivatives bearing a perfluorophenyl azide cross-linker at nucleotides in definite positions relative to the adenine of the AUG start codon. We found that the modifier at the IRES position -3 cross-links to ribosomal proteins uS11 and eS26. These proteins have been identified together with uS7 as those interacting with the mRNA nucleotide in position −3 relative to the first nucleotide of the codon directed to the P site by a cognate tRNA. Thus, our results indicate a certain difference in the locations of the above parts of HCV IRES and canonical mRNAs on 40S subunits. The modifier at the IRES positions +4/5 was attached to uS19, which is specific for ribosomal complexes with the P site tRNA and similar derivatives of model canonical mRNAs when the modifier is in the same positions. However, the cross-linking efficiency of the IRES derivative was drastically lower than that previously observed with derivatives of model mRNAs. This implies that the IRES ORF portion is correctly loaded into the mRNA binding channel only in a tiny fraction of the binary complexes.
Cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis Biochimie (IF 3.112) Pub Date : 2018-03-01 Jinyu Chi, Lei Wang, Xiaohui Zhang, Yu Fu, Yue Liu, Wenjia Chen, Wenxiu Liu, Zhiyu Shi, Xinhua Yin
Exercise training impacts exercise tolerance and bioenergetics in gastrocnemius muscle of non-obese type-2 diabetic Goto-Kakizaki rat in vivo Biochimie (IF 3.112) Pub Date : 2018-02-28 Michael Macia, Emilie Pecchi, Martine Desrois, Carole Lan, Christophe Vilmen, Bernard Portha, Monique Bernard, David Bendahan, Benoît Giannesini
The functional and bioenergetics impact of regular physical activity upon type-2 diabetic skeletal muscle independently of confounding factors of overweight remains undocumented. Here, gastrocnemius muscle energy fluxes, mitochondrial capacity and mechanical performance were assessed noninvasively and longitudinally in non-obese diabetic Goto-Kakizaki rats using magnetic resonance (MR) imaging and dynamic 31-phosphorus MR spectroscopy (31P-MRS) throughout a 6-min fatiguing bout of exercise performed before, in the middle (4-week) and at the end of an 8-week training protocol consisting in 60-min daily run on a treadmill. The training protocol reduced plasmatic insulin level (-61%) whereas blood glucose and non-esterified fatty acids levels remained unaffected, thereby indicating an improvement of insulin sensitivity. It also increased muscle mitochondrial citrate synthase activity (+45%) but this increase did not enhance oxidative ATP synthesis capacity in working muscle in vivo while glycolytic ATP production was increased (+33%). On the other hand, the training protocol impaired maximal force-generating capacity (-9%), total amount of force produced (-12%) and increased ATP cost of contraction (+32%) during the fatiguing exercise. Importantly, these deleterious effects were transiently worsened in the middle of the 8-week period, in association with reduced oxidative capacity and increased basal [Pi]/[PCr] ratio (an in vivo biomarker of muscle damage). These data demonstrate that the beneficial effect of regular training on insulin sensitivity in non-obese diabetic rat occurs separately from any improvement in muscle mitochondrial function and might be linked to an increased capacity for metabolizing glucose through anaerobic process in exercising muscle.
A synergistic effect of phosphate, pH and Phe159 substitution on the formycin A association to the E. coli purine nucleoside phosphorylase Biochimie (IF 3.112) Pub Date : 2018-02-28 Małgorzata Prokopowicz, Joanna Cieśla, Borys Kierdaszuk
Inhibition of nuclear translocation of notch intracellular domain (NICD) by diosgenin prevented atherosclerotic Biochimie (IF 3.112) Pub Date : 2018-02-24 Ambika Binesh, Sivasithambaram Niranjali Devaraj, Halagowder Devaraj
Notch signaling plays a pivotal role in homeostasis and cardiovascular development. The role of Notch signaling in atherosclerosis cannot be complete without analysing the key role of Notch in macrophages, which trigger the inflammatory response and subsequent plaque formation in atherosclerosis. Diosgenin showed its anti-atherosclerotic property by the unifying mechanism of suppressing the expression of Notch Signaling pathway, particularly the nuclear translocation of notch intracellular domain (NICD) in aorta and in differentiated macrophage cells. It is further confirmed by the inhibition of NICD by DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), which also restricted the differentiation of macrophage. Hence, inhibition of nuclear translocation of NICD by diosgenin aids in preventing atherosclerosis.
Structural and biophysical insight into dual site binding of the protoberberine alkaloid palmatine to parallel G-quadruplex DNA using NMR, fluorescence and circular dichroism spectroscopy Biochimie (IF 3.112) Pub Date : 2018-02-21 Kumar Padmapriya, Ritu Barthwal
Profile of CYP19A1 mRNA expression and aromatase activity during syncytialization of primary human villous trophoblast cells at term Biochimie (IF 3.112) Pub Date : 2018-02-21 Andrée-Anne Hudon Thibeault, Cathy Vaillancourt, J. Thomas Sanderson
Estrogen production by the human villous trophoblast is dependent on the biosynthetic enzyme aromatase (CYP19; CYP19A1) and is crucial for successful placental development and pregnancy outcome. Using villous cytotrophoblast cells (vCTs) freshly isolated from normal term placenta, we characterized the promoter-specific expression of CYP19A1 mRNA (derived from promoters I.1, I.4, I.8 or total transcript) and aromatase activity during villous trophoblast syncytialization. CYP19A1 mRNA levels and aromatase activity in vCTs reached a maximum after about 48 h of culture. The cAMP inducer forskolin (10 μM) and protein kinase C stimulant phorbol myristate acetate (1 μM) increased CYP19A1 mRNA levels by 1.8- and 1.6-fold, respectively, as well as inducing aromatase catalytic activity. Dexamethasone (100 nM) and vascular endothelial growth factor (5 ng/mL) decreased CYP19A1 mRNA levels, while having no effect on aromatase activity. Our results emphasize the importance of not solely studying CYP19A1 regulation and function at the mRNA level but also by considering posttranslational mechanisms that alter the final catalytic activity of aromatase.
Activated CXCL5-CXCR2 axis promotes the migration, invasion and EMT of papillary thyroid carcinoma cells via modulation of β-catenin pathway Biochimie (IF 3.112) Pub Date : 2018-02-20 Dong Cui, Yongfu Zhao, Jingchao Xu
Initiation of epithelial-to-mesenchymal transition (EMT) is common in papillary thyroid carcinoma (PTC) and may contribute to its metastasis. Aims of the present study are to investigate whether and how the C-X-C motif chemokine ligand (CXCL)-5/C-X-C motif receptor 2 (CXCR2) axis affects PTC metastasis, with a focus on the EMT process. Herein, two PTC cell lines, KTC-1 and B-CPAP cells, identified as CXCR2-positive cells were used as the cell model. We found that a 24-h stimulation of 1 or 10 nM recombinant human CXCL5 (rhCXCL5) enhanced the migration and invasion of both KTC-1 and B-CPAP cells without affecting their proliferation. The migration- and invasion-promoting effects of rhCXCL5 were attenuated if CXCR2 was silenced by its specific short hairpin RNAs (shRNAs). EMT initiation is defined as downregulation of epithelial-cadherin (E-cadherin) and upregulation of N-cadherin, Vimentin and Snail. Our data showed that rhCXCL5-induced EMT in PTC cells was suppressed by CXCR2 shRNA. Furthermore, the active CXCL5-CXCR2 axis enhanced the phosphorylation of Akt at Ser 473 residue and that of glycogen synthase kinase-3 (GSK-3β) at Ser 9 residue, and accelerated the nuclear accumulation of β-catenin in PTC cells. Re-expression of the active form of β-catenin in PTC cells rescued their impaired invasiveness caused by the blockade of CXCL5-CXCR2 axis. In addition, CXCL5 and CXCR2 were overexpressed in the metastatic lymph nodes obtained from 18 patients with PTC. In summary, our study demonstrates that the activated CXCL5-CXCR2 axis contributes to the metastatic phenotype of PTC cells by modulating Akt/GSK-3β/β-catenin pathway.
Modulation of Kv3.1b potassium channel level and intracellular potassium concentration in 158N murine oligodendrocytes and BV-2 murine microglial cells treated with 7-ketocholesterol, 24S-hydroxycholesterol or tetracosanoic acid (C24:0) Biochimie (IF 3.112) Pub Date : 2018-02-17 Maryem Bezine, Sonia Maatoug, Rym Ben Khalifa, Meryam Debbabi, Amira Zarrouk, Yuqin Wang, William J. Griffiths, Thomas Nury, Mohammad Samadi, Anne Vejux, Jérôme de Sèze, Thibault Moreau, Riadh Kharrat, Mohamed El Ayeb, Gérard Lizard
Structural and biophysical insight into dual site binding of the protoberberine alkaloid palmatine to parallel G-quadruplex DNA using NMR, fluorescence and Circular Dichroism spectroscopy Biochimie (IF 3.112) Pub Date : 2018-02-17 Kumar Padmapriya, Ritu Barthwal
Plant derived small molecules, which interact with and stabilize G-quadruplex DNA, act as inhibitors of telomere elongation and oncogene expression in humans. The inhibition of telomerase enzyme has immense potential since it is over expressed in most cancer cells. Interaction of palmatine, an antitumor alkaloid, to parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)]4, has been investigated using Nuclear Magnetic Resonance (NMR), fluorescence and Circular Dichroism (CD) spectroscopy. Titrations were monitored by 1H and 31P NMR spectra and solution structure of palmatine-[d(TTGGGGT)]4 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by CD, 1H NMR and Differential Scanning Calorimetry (DSC). Binding of palmatine induces 98% enhancement of fluorescence accompanied by blue shift ∼8 nm. CD spectral bands of DNA show minor changes. Diffusion NMR studies confirm formation of a stable complex. Proton NMR signals of palmatine shift upfield upon binding and NOE cross peaks of H10, H3, H28, 5OCH3 protons with T2, A3/G3, G6 and T7 residues reveal dual recognition sites in both G-quadruplex DNA sequences, resulting in thermal stabilization of G-quadruplex by ∼13-17 °C. Restrained molecular dynamics simulations using NOE distance restraints for 2:1 palmatine-[d(TTGGGGT)]4 complex reveal end-stacking of palmatine at G6pT7 step and groove binding along T2pG3 step. Binding to [d(TTAGGGT)]4 takes place at T2pA3pG4 and G6pT7 steps. Structural features of molecular recognition of two different G-quadruplex DNA sequences by palmatine have relevance in rational drug development for anti-cancer therapy.
Temozolomide Affects Extracellular Vesicles Released by Glioblastoma Cells Biochimie (IF 3.112) Pub Date : 2018-02-15 Gwennan André-Grégoire, Nicolas Bidère, Julie Gavard
Glioblastoma multiforme (GBM) is the most aggressive primary tumour within the brain as well as the most common and lethal cerebral cancer, mainly because of treatment failure. Indeed, tumour recurrence is inevitable and fatal in a short period of time. Glioblastoma stem-like cells (GSCs) are thought to participate in tumour initiation, expansion, resistance to treatments, including to the alkylating chemotherapeutic agent temozolomide, and relapse. Here, we assessed whether extracellular vesicles (EVs) released by GSCs could disseminate factors involved in resistance mechanisms. We first characterized EVs either circulating in peripheral blood from newly diagnosed patients or released by patient-derived temozolomide-resistant GSCs. We found that EVs from both sources were mainly composed of particles homogeneous in size (50-100 nm), while they were more abundant in liquid biopsies from GBM patients, as compared to healthy donors. Further, mass spectrometry analysis from GSC-derived EVs unveiled that particles from control and temozolomide-treated cells share core components of EVs, as well as ribosome- and proteasome-associated networks. More strikingly, temozolomide treatment led to the enrichment of EVs with cargoes dedicated to cell adhesion processes. Thus, while relatively inefficient in killing GSCs in vitro, temozolomide could instead increase the release of pro-tumoral information.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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