BBA - General Subjects (v.1840, #9)
Editorial Board (i).
Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: A comparative proteomic analysis by Li-Chen Ge; Zhuo-Jia Chen; Hao Liu; Kun-Shui Zhang; Qiao Su; Xiang-Yu Ma; Hong-Bin Huang; Zhen-Dong Zhao; Yu-Ye Wang; John P. Giesy; Jun Du; Hong-Sheng Wang (2663-2673).
Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study.We utilized proteomic study to indentify the protein expression changes of Sertoli TM4 cells treated with 10− 8 M and 10− 5 M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis.Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10− 8 M and 10− 5 M BPA, respectively, for 24 h. Exposure to 10− 5 M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10− 8 M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages.Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism.Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.Our study demonstrates, for the first time, that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation of cell by promoting energy metabolism.Display Omitted
Keywords: Proteomics; Proliferation; TM4 cell; Oxidative stress; Energy metabolism; In vitro;
The Broad Complex isoform 2 (BrC-Z2) transcriptional factor plays a critical role in vitellogenin transcription in the silkworm Bombyx mori by Congwen Yang; Ying Lin; Hongling Liu; Guanwang Shen; Juan Luo; Haiyan Zhang; Zhixin Peng; Enxiang Chen; Runmiao Xing; Chaoshan Han; Qingyou Xia (2674-2684).
Vitellogenin (Vg) is synthesized in the fat body of the female silkworm Bombyx mori and transported to the oocyte as a source of nutrition for embryo development. It is well known that ecdysone regulates physiological, developmental and behavioral events in silkworm. However, it is still not clear how the ecdysone regulates B. mori Vg (BmVg) transcription.Electrophoretic mobility shift assay (EMSA) and cell transfection assay were used to reveal whether BmBrC-Z2 is involved in regulating BmVg transcription. RNAi was employed to illustrate the function of BmBrC-Z2 in the silkworm egg formation and development.(1) The transcription of BmVg can be induced by ecdysone in the female fat body. (2) Three putative BrC-Z2 cis-response elements were mapped to regions flanking the BmVg gene. (3) BmBrC-Z2 required direct binding to the cis-response elements on the BmVg promoter. (4) Over-expression of three BmBrC isoforms in the cell line showed that only BmBrC-Z2 could induce the BmVg promoter activity. (5) RNA interference (RNAi) of BmBrC-Z2 in female remarkably reduced BmVg synthesis and led to destructive affection on egg formation. The dsRNA of BmBrC-Z2 treated moths laid fewer and whiter eggs compared to the control.BmBrC-Z2 transported the ecdysone signal then regulated BmVg transcription directly to control vitellogenesis and egg formation in the silkworm.The results of this study revealed that BmBrC-Z2 as a key factor to mediate ecdysone regulates reproduction in the silkworm.
Keywords: Ecdysone; Broad Complex; Vitellogenin; Bombyx mori;
Mutagenesis and molecular dynamics simulations revealed the chitooligosaccharide entry and exit points for chitinase D from Serratia proteamaculans by Jogi Madhuprakash; Karunakar Tanneeru; Bhavana Karlapudi; Lalitha Guruprasad; Appa Rao Podile (2685-2694).
Transglycosylation (TG) activity is a property of glycosyl hydrolases (GHs) with which new glycosidic bonds are introduced between donor and acceptor sugar molecules. This special property of the GHs has potential to generate longer chain chitooligosaccharides (CHOS) that show elicitor activity in plants. We hypothesize that TG activity could be improved by retaining the substrate for a longer duration in the catalytic site.Four variants of chitinase D from Serratia proteamaculans (SpChiD) i.e. G119S, G119W, W120A and G201W were analyzed in detail for improved TG activity using high performance liquid chromatography (HPLC) and high resolution mass spectrometry (HRMS). The results were strongly supported by 50 ns molecular dynamics (MD) simulations and estimated solvated interaction energies (SIE).The mutant G119W lost much of both hydrolytic and TG activities, while the mutant G201W displayed increased TG. The trajectory of MD simulations of the mutant G119W showed that the indole rings of two adjacent Trp residues create a major hindrance for the DP4 movement towards the catalytic center. Increased van der Waals (vdW) and coulombic interactions between DP4 substrate and the Trp-201 resulted in enhanced TG activity with the mutant G201W. The average number of hydrogen bonds observed for the DP4 substrate was increased for the mutants G119W and G201W compared to SpChiD.The increase in TG activity could be due to partial blocking of product exit of SpChiD.This new approach can be used for generating mutants of GHs with improved TG activity to produce longer chain oligosaccharides.Display Omitted
Keywords: Chitinases; Chitooligosaccharides; SpChiD; Transglycosylation; Mutagenesis; Molecular Dynamics Simulations;
Binding of [Cr(phen)3]3+ to transferrin at extracellular and endosomal pHs: Potential application in photodynamic therapy by Pablo F. Garcia; Judith Toneatto; María Jazmín Silvero; Gerardo A. Argüello (2695-2701).
Transferrin is an iron-binding blood plasma glycoprotein that controls the level of free iron in biological fluids. This protein has been deeply studied in the past few years because of its potential use as a strategy of drug targeting to tumor tissues. Chromium complex, [Cr(phen)3]3+ (phen = 1,10-phenanthroline), has been proposed as photosensitizers for photodynamic therapy (PDT). Thus, we analyzed the binding of chromium complex, [Cr(phen)3]3+, to transferrin for a potential delivery of this diimine complex to tumor cells for PDT.The interaction between [Cr(phen)3]3+ and holotransferrin (holoTf) was studied by fluorescence quenching technique, circular dichroism (CD) and ultraviolet (UV)–visible spectroscopy.[Cr(phen)3]3+ binds strongly to holoTf with a binding constant around 105 M−1, that depends on the pH. The thermodynamic parameters indicated that hydrophobic interactions played a major role in the binding processes. The CD studies showed that there are no conformational changes in the secondary and tertiary structures of the protein.These results suggest that the binding process would occur in a site different from the specific iron binding sites of the protein and would be the same in both protein states. As secondary and tertiary structures of transferrin do not show remarkable changes, we propose that the TfR could recognize the holoTf despite having a chromium complex associated.Understanding the interaction between [Cr(phen)3]3+ with transferrin is relevant because this protein could be a delivery agent of Cr(III) complex to tumor cells. This can allow us to understand further the role of Cr(III) complex as sensitizer in PDT.
Keywords: Tris(1,10-phenanthroline)chromium(III); Apotransferrin; Holotransferrin; Binding; Drug delivery; Photodynamic therapy;
Modification of extracorporeal photopheresis technology with porphyrin precursors. Comparison between 8-methoxypsoralen and hexaminolevulinate in killing human T-cell lymphoma cell lines in vitro by B. Čunderlíková; V. Vasovič; L.L. Randeberg; E. Christensen; T. Warloe; J.M. Nesland; Q. Peng (2702-2708).
Extracorporeal photopheresis that exposes isolated white blood cells to 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light is used for the management of cutaneous T-cell lymphoma and graft-versus-host disease. 8-MOP binds to DNA of both tumor and normal cells, thus increasing the risk of carcinogenesis of normal cells; and also kills both tumor and normal cells with no selectivity after UV-A irradiation. Hexaminolevulinate (HAL)-induced protoporphyrin-IX is a potent photosensitizer that localizes at membranous structures outside of the nucleus of a cell. HAL-mediated photodynamic therapy selectively destroys activated/transformed lymphocytes and induces systemic anti-tumor immunity. The aim of the present study was to explore the possibility of using HAL instead of 8-MOP to kill cells after UV-A exposure.Human T-cell lymphoma Jurkat and Karpas 299 cell lines were used to evaluate cell photoinactivation after 8-MOP and/or HAL plus UV-A light with cell proliferation and long term survival assays. The mode of cell death was also analyzed by fluorescence microscopy.Cell proliferation was decreased by HAL/UV-A, 8-MOP/UV-A or HAL/8-MOP/UV-A. At sufficient doses, the cells were killed by all the regimens; however, the mode of cell death was dependent on the treatment conditions. 8-MOP/UV-A produced apoptotic death exclusively; whereas both apoptosis and necrosis were induced by HAL/UV-A.8-MOP can be replaced by HAL to inactivate the Jurkat and Karpas 299 T-cell lymphoma cells after UV-A irradiation via apoptosis and necrosis. This finding may have an impact on improved efficacy of photopheresis.
Keywords: Hexaminolevulinate; Photodynamic therapy; Extracorporeal photopheresis; Protoporphyrin IX; Ultraviolet-A light; Psoralen;
Diabetes, oxidative stress and therapeutic strategies by Luc Rochette; Marianne Zeller; Yves Cottin; Catherine Vergely (2709-2729).
Diabetes has emerged as a major threat to health worldwide.The exact mechanisms underlying the disease are unknown; however, there is growing evidence that excess generation of reactive oxygen species (ROS), largely due to hyperglycemia, causes oxidative stress in a variety of tissues. Oxidative stress results from either an increase in free radical production, or a decrease in endogenous antioxidant defenses, or both. ROS and reactive nitrogen species (RNS) are products of cellular metabolism and are well recognized for their dual role as both deleterious and beneficial species. In type 2 diabetic patients, oxidative stress is closely associated with chronic inflammation. Multiple signaling pathways contribute to the adverse effects of glucotoxicity on cellular functions. There are many endogenous factors (antioxidants, vitamins, antioxidant enzymes, metal ion chelators) that can serve as endogenous modulators of the production and action of ROS. Clinical trials that investigated the effect of antioxidant vitamins on the progression of diabetic complications gave negative or inconclusive results. This lack of efficacy might also result from the fact that they were administered at a time when irreversible alterations in the redox status are already under way. Another strategy to modulate oxidative stress is to exploit the pleiotropic properties of drugs directed primarily at other targets and thus acting as indirect antioxidants.It appears important to develop new compounds that target key vascular ROS producing enzymes and mimic endogenous antioxidants.This strategy might prove clinically relevant in preventing the development and/or retarding the progression of diabetes associated with vascular diseases.
Keywords: Diabetes; Oxidative stress; Reactive oxygen specie; Signaling pathway; Therapeutics;
Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated chitosan nanoparticles towards colon cancer treatment by A. Anitha; N. Deepa; K.P. Chennazhi; Vinoth-Kumar Lakshmanan; R. Jayakumar (2730-2743).
Evaluation of the combinatorial anticancer effects of curcumin/5-fluorouracil loaded thiolated chitosan nanoparticles (CRC-TCS-NPs/5-FU-TCS-NPs) on colon cancer cells and the analysis of pharmacokinetics and biodistribution of CRC-TCS-NPs/5-FU-TCS-NPs in a mouse model.CRC-TCS-NPs/5-FU-TCS-NPs were developed by ionic cross-linking. The in vitro combinatorial anticancer effect of the nanomedicine was proven by different assays. Further the pharmacokinetics and biodistribution analyses were performed in Swiss Albino mouse using HPLC.The 5-FU-TCS-NPs (size: 150 ± 40 nm, zeta potential: + 48.2 ± 5 mV) and CRC-TCS-NPs (size: 150 ± 20 nm, zeta potential: + 35.7 ± 3 mV) were proven to be compatible with blood. The in vitro drug release studies at pH 4.5 and 7.4 showed a sustained release profile over a period of 4 days, where both the systems exhibited a higher release in acidic pH. The in vitro combinatorial anticancer effects in colon cancer (HT29) cells using MTT, live/dead, mitochondrial membrane potential and cell cycle analysis measurements confirmed the enhanced anticancer effects (2.5 to 3 fold). The pharmacokinetic studies confirmed the improved plasma concentrations of 5-FU and CRC up to 72 h, unlike bare CRC and 5-FU.To conclude, the combination of 5-FU-TCS-NPs and CRC-TCS-NPs showed enhanced anticancer effects on colon cancer cells in vitro and improved the bioavailability of the drugs in vivo.The enhanced anticancer effects of combinatorial nanomedicine are advantageous in terms of reduction in the dosage of 5-FU, thereby improving the chemotherapeutic efficacy and patient compliance of colorectal cancer cases.Display Omitted
Keywords: Combinatorial nanomedicine; Colon cancer; Pharmacokinetics; Thiolated chitosan; 5-Fluorouracil; Curcumin;
Interaction of the scorpion toxin discrepin with Kv4.3 channels and A-type K+ channels in cerebellum granular cells by Cristiana Picco; Gerardo Corzo; Lourival D. Possani; Gianfranco Prestipino (2744-2751).
The peptide discrepin from the α-KTx15 subfamily of scorpion toxins preferentially affects transient A-type potassium currents, which regulate many aspects of neuronal function in the central nervous system. However, the specific Kv channel targeted by discrepin and the molecular mechanism of interaction are still unknown.Different variant peptides of discrepin were chemically synthesized and their effects were studied using patch clamp technique on rat cerebellum granular cells (CGC) and HEK cells transiently expressing Kv4.3 channels.Functional analysis indicated that nanomolar concentrations of native discrepin blocked Kv4.3 expressed channels, as previously observed in CGC. Similarly, the apparent affinities of all mutated peptides for Kv4.3 expressed channels were analogous to those found in CGC. In particular, in the double variant [V6K, D20K] the apparent affinity increased about 10-fold, whereas in variants carrying a deletion (ΔK13) or substitution (K13A) at position K13, the blockage was removed and the apparent affinity decreased more than 20-fold.These results indicate that Kv4.3 is likely the target of discrepin and highlight the importance of the basic residue K13, located in the α-helix of the toxin, for current blockage.We report the first example of a Kv4 subfamily potassium channel blocked by discrepin and identify the amino acid residues responsible for the blockage. The availability of discrepin variant peptides stimulates further research on the functions and pharmacology of neuronal Kv4 channels and on their possible roles in neurodegenerative disorders.
Keywords: A-type currents; Discrepin; Heterologous expression; K+ channel; Scorpion toxin;
Sialosignaling: Sialyltransferases as engines of self-fueling loops in cancer progression by Fabio Dall'Olio; Nadia Malagolini; Marco Trinchera; Mariella Chiricolo (2752-2764).
Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression.To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression.We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen–Friedenreich-associated antigens, sialyl Lewis antigens, α2,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information.Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer.
Keywords: Glycosylation; Sialylation; Gangliosides; Neural cell adhesion molecule; Integrins; Chemoresistance;
Synthetic peptides derived from the C-terminal 6 kDa region of Plasmodium falciparum SERA5 inhibit the enzyme activity and malaria parasite development by Shivani Kanodia; Gautam Kumar; Luca Rizzi; Alessandro Pedretti; Anthony N. Hodder; Sergio Romeo; Pawan Malhotra (2765-2775).
Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site.In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential.The final proteolytic step of PfSERA5 involves removal of a C-terminal ~ 6 kDa fragment that results in the generation of a catalytically active ~ 50 kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~ 6 kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5.Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress.These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~ 6 kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.Display Omitted
Keywords: Peptide synthesis; Site directed mutagenesis; Enzyme kinetics; Plasmodium;
Metal-mediated oxidative DNA damage induced by methylene blue by Yusuke Hiraku; Hiroyuki Goto; Masaki Kohno; Shosuke Kawanishi; Mariko Murata (2776-2782).
Methylene blue (MB) is used for various clinical purposes, including chromoendoscopy and methemoglobinemia treatment. However, MB induces tumors of pancreatic islets and small intestine in experimental animals. This finding raises a possibility that MB induces carcinogenicity in these organs via light-independent mechanisms, although MB is known to cause light-dependent DNA damage.We investigated the mechanism of MB-induced DNA damage using 32P-5′-end-labeled DNA fragments of human tumor-relevant genes. We investigated the redox reaction of MB by UV–visible spectrometry.MB induced DNA damage at the 5′-ACG-3′ sequence, a hot spot of the p53 gene, in the presence of NADH and Cu(II). DNA damage was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator. MB induced DNA damage at every nucleotide in the presence of NADH and Fe(III)-ethylenediaminetetraacetic acid, which was inhibited by •OH scavengers and catalase. MB significantly increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, an oxidative DNA lesion, in the presence of NADH and metal ions. UV–visible spectrometry revealed that the absorbance of oxidized form of MB at 668 nm was decreased by NADH, and the addition of metal ions attenuated the spectral change.MB undergoes NADH-dependent reduction followed by metal ion-mediated reoxidation. Reduced metal ions [Cu(I) and Fe(II)] interact with H2O2, generated during the redox reaction, to produce Cu(I)OOH and •OH that cause DNA damage, respectively. These findings suggest that metal-mediated DNA damage contributes to MB-mediated carcinogenesis.This study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.
Keywords: Methylene blue; DNA damage; Metal ion; Reactive oxygen species; NADH; Carcinogenesis;
Biogenesis and adhesion of type 1 and P pili by James Lillington; Sebastian Geibel; Gabriel Waksman (2783-2793).
Uropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) in approximately 50% of women. These bacteria use type 1 and P pili for host recognition and attachment. These pili are assembled by the chaperone-usher pathway of pilus biogenesis.The review examines the biogenesis and adhesion of the UPEC type 1 and P pili. Particular emphasis is drawn to the role of the outer membrane usher protein. The structural properties of the complete pilus are also examined to highlight the strength and functionality of the final assembly.The usher orchestrates the sequential addition of pilus subunits in a defined order. This process follows a subunit-incorporation cycle which consists of four steps: recruitment at the usher N-terminal domain, donor-strand exchange with the previously assembled subunit, transfer to the usher C-terminal domains and translocation of the nascent pilus.Adhesion by the type 1 and P pili is strengthened by the quaternary structure of their rod sections. The rod is endowed with spring-like properties which provide mechanical resistance against urine flow. The distal adhesins operate differently from one another, targeting receptors in a specific manner.The biogenesis and adhesion of type 1 and P pili are being therapeutically targeted, and efforts to prevent pilus growth or adherence are described.The combination of structural and biochemical study has led to the detailed mechanistic understanding of this membrane spanning nano-machine. This can now be exploited to design novel drugs able to inhibit virulence. This is vital in the present era of resurgent antibiotic resistance. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
Keywords: Chaperone; Usher; Pilus; Donor-strand exchange; Pilicides; Macromolecular machine;
Heteroglucan-dendrimer glycoconjugate: a modulated construct with augmented immune responses and signaling phenomena by K. Sanjana P. Devi; Birendra Behera; Banalata Sahoo; Tapas K. Maiti (2794-2805).
Newer strategies for augmenting immune responses of pharmacologically active glucans may serve to improve the medicinal potential of these biomolecules. With this aim, the present work was focused on generating targeted high molecular size glucan particles with magnified immune response activity.Heteroglucans were conjugated with PAMAM dendrimers using a Schiff base reductive amination reaction to generate a polytethered molecule with multiple glucan motifs. The modulated construct was characterized by FTIR, TEM, 1H NMR and dynamic light scattering (DLS) methods. Effects of conjugated glucans were examined in RAW 264.7 macrophage cells as well as in S-180 murine tumor models.Dendrimer-conjugated glucans were found to exhibit a two-fold increase in immune stimulation in comparison to unconjugated glucans. This may be corroborated by the predominant enhancement in immunological functions such as nitric oxide production, ROS generation and immune directed tumor inhibition in murine models. Immune cell surface markers (CD4, CD8, CD19, MHC-II) and cytokine levels were also found to be highly up-regulated in the splenocytes of mice subjected to particulate glucan administration. Our study also demonstrated that conjugated glucan treatment to RAW 264.7 cells strongly enhanced the phosphorylation of two downstream signalling molecules of the mitogen activated protein kinase (MAPKs) family: p38 and MEK1/2 relative to single glucans thereby relating molecular mechanisms with enhanced immune stimulation.The results obtained thus support that particulate format of soluble heteroglucan will thereby improve its functionality and identify leads in therapeutic competence.Display Omitted
Keywords: Dendrimer conjugated heteroglucan; Immune stimulation; Tumor inhibition; Signalling; Biomolecules; Therapeutic;
N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-l-tryptophan by Yousuke Kouno; Makoto Anraku; Keishi Yamasaki; Yoshiro Okayama; Daisuke Iohara; Yu Ishima; Toru Maruyama; Ulrich Kragh-Hansen; Fumitoshi Hirayama; Masaki Otagiri (2806-2812).
Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products.Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4 weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration.N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present.The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations.N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.Display Omitted
Keywords: Human serum albumin; Photo-oxidation; Antioxidant activity; Octanoate; N-Acetyl-l-tryptophan; N-Acetyl-l-methionine;
The cytochrome P450 genes of channel catfish: Their involvement in disease defense responses as revealed by meta-analysis of RNA-Seq data sets by Jiaren Zhang; Jun Yao; Ruijia Wang; Yu Zhang; Shikai Liu; Luyang Sun; Yanliang Jiang; Jianbin Feng; Nannan Liu; David Nelson; Geoff Waldbieser; Zhanjiang Liu (2813-2828).
Cytochrome P450s (CYPs) encode one of the most diverse enzyme superfamily in nature. They catalyze oxidative reactions of endogenous molecules and exogenous chemicals.We identified CYPs genes through in silico analysis using EST, RNA-Seq and genome databases of channel catfish. Phylogenetic analyses and conserved syntenic analyses were conducted to determine their identities and orthologies. Meta-analysis of RNA-Seq databases was conducted to analyze expression profile of CYP genes following bacterial infection.A full set of 61 CYP genes was identified and characterized in channel catfish. Phylogenetic tree and conserved synteny provided strong evidence of their identities and orthorlogy. Lineage-specific gene duplication was evident in a number of clans in channel catfish. CYP46A1 is missing in the catfish genome as observed with syntenic analysis and RT-PCR analysis. Thirty CYPs were found up- or down-regulated in liver, while seven and eight CYPs were observed regulated in intestine and gill following bacterial infection.We systematically identified and characterized a full set of 61 CYP genes in channel catfish and studied their expression profiles after bacterial infection. While bacterial challenge altered the expression of large numbers of CYP genes, the mechanisms and significance of these changes are not known.This work provides an example to systematically study CYP genes in non-model species. Moreover, it provides a basis for further toxicological and physiological studies in channel catfish.
Keywords: Cytochrome P450; CYP; Catfish; Genome; Immunity; Bacterial infection;
Roles of carbonic anhydrase 8 in neuronal cells and zebrafish by Min-Syuan Huang; Tze-Kai Wang; Yi-Wen Liu; Yi-Ting Li; Tang-Hao Chi; Chih-Wei Chou; Mingli Hsieh (2829-2842).
Carbonic anhydrase 8 (CA8) is an isozyme of α-carbonic anhydrases (CAs). Previous studies showed that CA8 can be detected in human adult brain, with more intense expression in the cerebellum. Single mutations in CA8 were reported to cause novel syndromes like ataxia, mild mental retardation or the predisposition to quadrupedal gait.In the present study, we examine the functions of CA8 in neuronal cell lines, mouse cerebellar granule neurons and zebrafish.We demonstrated that overexpression of CA8 in neuronal cells significantly decreased cell death under staurosporine treatment. Moreover, CA8 overexpression significantly increased cell migration and invasion ability in neuronal cells and in mouse cerebellar granule neurons, implicating that CA8 may be involved in neuron motility and oncogenesis. By using zebrafish as an animal model, motor reflection of 3 dpf zebrafish embryos was significantly affected after the down-regulation of CA8 through ca8 morpholino.We concluded that CA8 overexpression desensitizes neuronal cells to STS induced apoptotic stress and increases cell migration and invasion ability in neuronal cells. In addition, down-regulated CA8 decreases neuron mobility in neuronal cells and leads to abnormal calcium release in cerebellar granule neurons. Knockdown of the ca8 gene results in an abnormal movement pattern in zebrafish.Our findings provide evidence to support that the impaired protective function of CA8 contributes to human neuropathology, and to suggest that zebrafish can be used as an animal model to study the biological functions of human CA8 in vivo.
Keywords: Carbonic anhydrase 8; SK-N-SH; Neuro-2a; Zebrafish; Cerebellar granule neuron; Cell migration and invasion;
Impact of protein and ligand impurities on ITC-derived protein–ligand thermodynamics by Stefan Grüner; Manuel Neeb; Luzi Jakob Barandun; Frank Sielaff; Christoph Hohn; Shun Kojima; Torsten Steinmetzer; François Diederich; Gerhard Klebe (2843-2850).
The thermodynamic characterization of protein–ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations.Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC. Data curation was attempted also considering information from analytical techniques to correct stoichiometry.We used trypsin and tRNA-guanine transglycosylase (TGT), together with high affinity ligands to investigate the effect of errors in protein concentration as well as the impact of ligand impurities on the apparent thermodynamics. We found that errors in protein concentration did not change the thermodynamic properties obtained significantly. However, most ligand impurities led to pronounced changes in binding enthalpy. If protein binding of the respective impurity is not expected, the actual ligand concentration was corrected for and the thus revised data compared to thermodynamic properties obtained with the respective pure ligand. Even in these cases, we observed differences in binding enthalpy of about 4 kJ ⋅ mol− 1, which is considered significant.Our results indicate that ligand purity is the critical parameter to monitor if accurate thermodynamic data of a protein–ligand complex are to be recorded. Furthermore, artificially changing fitting parameters to obtain a sound interaction stoichiometry in the presence of uncharacterized ligand impurities may lead to thermodynamic parameters significantly deviating from the accurate thermodynamic signature.Display Omitted
Keywords: Isothermal titration calorimetry; Impurities; Enthalpy; Gibbs free energy; Dynamic light scattering;
Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA by Varahenage R. Perera; Gerald L. Newton; Jonathan M. Parnell; Elizabeth A. Komives; Kit Pogliano (2851-2861).
Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins.We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays.BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity.BstA is a bacillithiol transferase from S. aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules.BstA is an active bacillithiol transferase from S. aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from B. subtilis YfiT.
Keywords: bacillithiol; bacillithiol transferase; detoxification; Staphylococcus aureus;
Intact and N- or C-terminal end truncated AQP0 function as open water channels and cell-to-cell adhesion proteins: End truncation could be a prelude for adjusting the refractive index of the lens to prevent spherical aberration by S. Sindhu Kumari; Kulandaiappan Varadaraj (2862-2877).
Investigate the impact of natural N- or C-terminal post-translational truncations of lens mature fiber cell Aquaporin 0 (AQP0) on water permeability (Pw) and cell-to-cell adhesion (CTCA) functions.The following deletions/truncations were created by site-directed mutagenesis (designations in parentheses): Amino acid residues (AA) 2–6 (AQP0-N-del-2-6), AA235–263 (AQP0-1-234), AA239–263 (AQP0-1-238), AA244–263 (AQP0-1-243), AA247–263 (AQP0-1-246), AA250–263 (AQP0-1-249) and AA260–263 (AQP0-1-259). Protein expression was studied using immunostaining, fluorescent tags and organelle-specific markers. Pw was tested by expressing the respective complementary ribonucleic acid (cRNA) in Xenopus oocytes and conducting osmotic swelling assay. CTCA was assessed by transfecting intact or mutant AQP0 into adhesion-deficient L-cells and performing cell aggregation and adhesion assays.AQP0-1-234 and AQP0-1-238 did not traffic to the plasma membrane. Trafficking of AQP0-N-del-2-6 and AQP0-1-243 was reduced causing decreased membrane Pw and CTCA. AQP0-1-246, AQP0-1-249 and AQP0-1-259 mutants trafficked properly and functioned normally. Pw and CTCA functions of the mutants were directly proportional to the respective amount of AQP0 expressed at the plasma membrane and remained comparable to those of intact AQP0 (AQP0-1-263).Post-translational truncation of N- or C-terminal end amino acids does not alter the basal water permeability of AQP0 or its adhesive functions. AQP0 may play a role in adjusting the refractive index to prevent spherical aberration in the constantly growing lens.Similar studies can be extended to other lens proteins which undergo post-translational truncations to find out how they assist the lens to maintain transparency and homeostasis for proper focusing of objects on to the retina.Display Omitted
Keywords: AQP0; Water permeability; Cell-to-cell adhesion; AQP0 N-/ C-terminal truncation; Refractive index; Spherical aberration;
Glucose starvation-induced turnover of the yeast glucose transporter Hxt1 by Adhiraj Roy; Yong-Bae Kim; Kyu Hong Cho; Jeong-Ho Kim (2878-2885).
The budding yeast Saccharomyces cerevisiae possesses multiple glucose transporters with different affinities for glucose that enable it to respond to a wide range of glucose concentrations. The steady-state levels of glucose transporters are regulated in response to changes in the availability of glucose. This study investigates the glucose regulation of the low affinity, high capacity glucose transporter Hxt1.Western blotting and confocal microscopy were performed to evaluate glucose regulation of the stability of Hxt1. Our results show that glucose starvation induces endocytosis and degradation of Hxt1 and that this event requires End3, a protein required for endocytosis, and the Doa4 deubiquitination enzyme. Mutational analysis of the lysine residues in the Hxt1 N-terminal domain demonstrates that the two lysine residues, K12 and K39, serve as the putative ubiquitin-acceptor sites by the Rsp5 ubiquitin ligase. We also demonstrate that inactivation of PKA (cAMP-dependent protein kinase A) is needed for Hxt1 turnover, implicating the role of the Ras/cAMP-PKA glucose signaling pathway in the stability of Hxt1.Hxt1, most useful when glucose is abundant, is internalized and degraded when glucose becomes depleted. Of note, the stability of Hxt1 is regulated by PKA, known as a positive regulator for glucose induction of HXT1 gene expression, demonstrating a dual role of PKA in regulation of Hxt1.
Keywords: Yeast; Glucose transporter; Hxt1; Endocytosis;
Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for treatment of Alzheimer's disease by Jinggong Liu; Jun Qiu; Mingxue Wang; Ling Wang; Lijuan Su; Jinbo Gao; Qiong Gu; Jun Xu; Shi-Liang Huang; Lian-Quan Gu; Zhi-Shu Huang; Ding Li (2886-2903).
Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that is characterized by dementia, cognitive impairment, and memory loss. Diverse factors are related to the development of AD, such as increased level of β-amyloid (Aβ), acetylcholine, metal ion deregulation, hyperphosphorylated tau protein, and oxidative stress.The following methods were used: organic syntheses of 1H-phenanthro[9,10-d]imidazole derivatives, inhibition of self-mediated and metal-induced Aβ 1–42 aggregation, inhibition studies for acetylcholinesterase and butyrylcholinesterase, anti-oxidation activity studies, CD, MTT assay, transmission electron microscopy, dot plot assay, gel electrophoresis, Western blot, and molecular docking studies.We synthesized and characterized a new type of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for AD treatment. Our results showed that most of these derivatives exhibited strong Aβ aggregation inhibitory activity. Compound 9g had 74% Aβ 1–42 aggregation inhibitory effect at 10 μM concentration with its IC50 value of 6.5 μM for self-induced Aβ 1–42 aggregation. This compound also showed good inhibition of metal-mediated (Cu2 + and Fe2 +) and acetylcholinesterase-induced Aβ 1–42 aggregation, as indicated by using thioflavin T assay, transmission electron microscopy, gel electrophoresis, and Western blot. Besides, compound 9g exhibited cholinesterase inhibitory activity, with its IC50 values of 0.86 μM and 0.51 μM for acetylcholinesterase and butyrylcholinesterase, respectively. In addition, compound 9g showed good anti-oxidation effect with oxygen radical absorbance capacity (ORAC) value of 2.29.Compound 9g was found to be a potent multi-target-directed agent for Alzheimer's disease.Compound 9g could become a lead compound for further development as a multi-target-directed agent for AD treatment.
Keywords: Alzheimer's disease; Imidazole derivative; Aβ aggregation; Cholinesterase; Anti-oxidation;
Glycan specificity of a testis-specific lectin chaperone calmegin and effects of hydrophobic interactions by Masafumi Sakono; Akira Seko; Yoichi Takeda; Jun-ichi Aikawa; Masakazu Hachisu; Akihiko Koizumi; Kohki Fujikawa; Yukishige Ito (2904-2913).
Testis-specific chaperone calmegin is required for the generation of normal spermatozoa. Calmegin is known to be a homologue of endoplasmic reticulum (ER) residing lectin chaperone calnexin. Although functional similarity between calnexin and calmegin has been predicted, detailed information concerned with substrate recognition by calmegin, such as glycan specificity, chaperone function and binding affinity, are obscure.In this study, biochemical properties of calmegin and calnexin were compared using synthetic glycans and glycosylated or non-glycosylated proteins as substrates.Whereas their amino acid sequences are quite similar to each other, a certain difference in secondary structures was indicated by circular dichroism (CD) spectrum. While both of them inhibited protein heat-aggregation to a similar extent, calnexin exhibited a higher ability to facilitate protein folding. Similarly to calnexin, calmegin preferentially recognizes monoglucosylated glycans such as Glc1Man9GlcNAc2 (G1M9). While the surface hydrophobicity of calmegin was higher than that of calnexin, calnexin showed stronger binding to substrate. We reasoned that lectin activity, in addition to hydrophobic interaction, contributes to this strong affinity between calnexin and substrate.Although their similarity in carbohydrate binding specificities is high, there seems to be some differences in the mode of substrate recognition between calmegin and calnexin.Properties of calmegin as a lectin-chaperone were revealed in comparison with calnexin.
Keywords: Lectin chaperone; Calnexin; Calmegin; Glycan; Hydrophobicity;
Monitoring the kinetics of CellTrace™ calcein red-orange AM intracellular accumulation with spatial intensity distribution analysis by Zahra Hamrang; Hayley J. McGlynn; David Clarke; Jeffrey Penny; Alain Pluen (2914-2923).
Routine black box approaches quantify fluorescence intensity to profile the uptake of fluorophores, providing limited insight into microscopic events. Spatial intensity distribution analysis has previously been reported to quantify oligomerisation and number of particles from selected regions and profile intracellular distributions of labelled moieties.In this study, the concentration and time-dependent behaviour of CellTrace™ calcein red-orange (AM) intracellular accumulation was examined in colorectal adenocarcinoma cell line and bovine aortic endothelial cells. Monolayers were subjected to fluorescence correlation spectroscopy, fluorescence intensity and SpIDA measurements to determine differences in the rate and extent of intracellular accumulation.Intracellular accumulation data derived from Spatial intensity distribution analysis were found to correlate with that of fluorescence correlation spectroscopy and fluorescence intensity profiles. The extent of intracellular accumulation was found to be time and concentration-dependent in both cell lines examined, with no significant differences in the rate of intracellular accumulation.Spatial intensity distribution analysis applied at ‘proof of concept’ level is a rapid and user-friendly tool that can be applied to the quantification of intracellular concentration and kinetics of fluorophore uptake.Confocal imaging as a routinely implemented tool for profiling fluorescently-labelled species is often under-exploited for yielding quantitative parameters.
Keywords: Confocal microscopy; Fluorescence correlation spectroscopy; Spatial intensity distribution analysis; Fluorescence;
Altered ubiquitin-proteasome system leads to neuronal cell death in a spontaneous obese rat model by S. Sreenivasa Reddy; Karnam Shruthi; V. Sudhakar Reddy; G. Raghu; P. Suryanarayana; N.V. Giridharan; G. Bhanuprakash Reddy (2924-2934).
Obesity is associated with various progressive age-related diseases, including neurological disorders. However, underlying molecular basis for increased risk of neurodegeneration in obesity is unknown. A suitable animal model would immensely help in understanding the obesity-linked neurological problems.A spontaneously developed obese rat (WNIN/Ob) which is highly vulnerable for a variety of degenerative diseases was isolated from the existing WNIN stock rats. Ultrastructure of neurons in the cerebral cortex of 12-month old obese rats was evaluated by transmission electron microscopy. qRT-PCR and immunoblotting of ubiquitin C-terminal hydrolases (UCHs), ubiquitin, proteasomal sub-units, markers of ER stress and apoptosis were performed in the cerebral cortex. Proteasome activity was assayed by fluorometric method. Immunohistochemistry was performed for mediators of apoptosis, which was further confirmed by TUNEL assay. These investigations were also carried in high-fat diet-induced obese rat model.Neurons in the cerebral cortex of 12-month obese rats showed swollen mitochondria, disrupted ER and degenerating axons, nucleus and finally neurons. Results showed altered UPS, existence of ER stress, up-regulation of apoptotic markers and apoptosis in the cerebral cortex of obese rats. It appears that UCHL-1 mediated apoptosis through stabilizing p53 might play a role in neuronal cell death in obese rat. Similar changes were observed in the brain of diet-induced obese WNIN rats.Altered UPS could be one of the underlying mechanisms for the neuronal cell death in obese conditions.This is the first report to highlight the role of altered UPS in neurodegeneration due to obesity.Display Omitted
Keywords: Obesity; Brain; Endoplasmic reticulum stress; Proteasome; Ubiquitin C-terminal hydrolase; Apoptosis;
Hydrolytic enzymes conjugated to quantum dots mostly retain whole catalytic activity by Aditya Iyer; Anil Chandra; Rajaram Swaminathan (2935-2943).
Tagging a luminescent quantum dot (QD) with a biological like enzyme (Enz) creates value-added entities like quantum dot–enzyme bioconjugates (QDEnzBio) that find utility as sensors to detect glucose or beacons to track enzymes in vivo. For such applications, it is imperative that the enzyme remains catalytically active while the quantum dot is luminescent in the bioconjugate. A critical feature that dictates this is the quantum dot–enzyme linkage chemistry. Previously such linkages have put constraints on polypeptide chain dynamics or hindered substrate diffusion to active site, seriously undermining enzyme catalytic activity. In this work we address this issue using avidin–biotin linkage chemistry together with a flexible spacer to conjugate enzyme to quantum dot.The catalytic activity of three biotinylated hydrolytic enzymes, namely, hen egg white lysozyme (HEWL), alkaline phosphatase (ALP) and acetylcholinesterase (AChE) was investigated post-conjugation to streptavidin linked quantum dot for multiple substrate concentrations and varying degrees of biotinylation.We demonstrate that all enzymes retain full catalytic activity in the quantum dot–enzyme bioconjugates in comparison to biotinylated enzyme alone. However, unlike alkaline phosphatase and acetylcholinesterase, the catalytic activity of hen egg white lysozyme was observed to be increasingly susceptible to ionic strength of medium with rising level of biotinylation. This susceptibility was attributed to arise from depletion of positive charge from lysine amino groups after biotinylation.We reasoned that avidin–biotin linkage in the presence of a flexible seven atom spacer between biotin and enzyme poses no constraints to enzyme structure/dynamics enabling retention of full enzyme activity.Overall our results demonstrate for the first time that streptavidin–biotin chemistry can yield quantum dot enzyme bioconjugates that retain full catalytic activity as native enzyme.Display Omitted
Keywords: Semiconductor nanocrystals; Hen egg white lysozyme; Alkaline phosphatase; Acetylcholinesterase; Enzyme catalytic activity; Luminescence;
Amyloid fibrils compared to peptide nanotubes by Matjaž Žganec; Eva Žerovnik (2944-2952).
Prefibrillar oligomeric states and amyloid fibrils of amyloid-forming proteins qualify as nanoparticles. We aim to predict what biophysical and biochemical properties they could share in common with better researched peptide nanotubes. We first describe what is known of amyloid fibrils and prefibrillar aggregates (oligomers and protofibrils): their structure, mechanisms of formation and putative mechanism of cytotoxicity. In distinction from other neuronal fibrillar constituents, amyloid fibrils are believed to cause pathology, however, some can also be functional. Second, we give a review of known biophysical properties of peptide nanotubes. Finally, we compare properties of these two macromolecular states side by side and discuss which measurements that have already been done with peptide nanotubes could be done with amyloid fibrils as well.
Keywords: Amyloid fibrils; oligomers; optical properties; Peptide nanotubes; Quantum confinement; Toxicity;
Nucleocytoplasmic transport under stress conditions and its role in HSP70 chaperone systems by Shingo Kose; Naoko Imamoto (2953-2960).
In eukaryotic cells, molecular trafficking between the nucleus and cytoplasm is a highly regulated process related to cellular homeostasis and cellular signaling. However, various cellular stresses induce the perturbation of conventional nucleocytoplasmic transport pathways, resulting in the nucleocytoplasmic redistribution of many functional proteins.We describe the recent insights into the mechanism and functions of nuclear import of cytosolic chaperone HSP70 under stress conditions and the cellular distribution and functions of its co-chaperones.Hikeshi mediates the nuclear import of the molecular chaperone HSP70. A few of the regulators of the HSP70 chaperone system also accumulate in the nucleus under heat stress conditions. These proteins function collaboratively to protect cells from stress-induced damage and aid in the recovery of cells from stress.Studies on the regulation of nucleocytoplasmic transport under several cellular stresses should provide new insights into the fundamental principles of protein homeostasis (proteostasis) in both compartments, the nucleus and cytoplasm.
Keywords: Nucleocytoplasmic transport; Importin; Cellular stress; Hikeshi; Molecular chaperone; HSP70;
An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis by Jana Gerstmeier; Christina Weinigel; Dagmar Barz; Oliver Werz; Ulrike Garscha (2961-2969).
Subcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.FLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca2 +-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB4. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.The cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.We present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.
Keywords: 5-Lipoxygenase; 5-Lipoxygenase-activating protein; HEK293 cells; MK886; Arachidonic acid; 12-Lipoxygenase;
Stabilization of VEGF G-quadruplex and inhibition of angiogenesis by quindoline derivatives by Yue Wu; Li-Peng Zan; Xiao-Dong Wang; Yu-Jing Lu; Tian-Miao Ou; Jing Lin; Zhi-Shu Huang; Lian-Quan Gu (2970-2977).
Angiogenesis is thought to be important in tumorigenesis and tumor progress. Vascular endothelial growth factor (VEGF) is a pluripotent cytokine and angiogenic growth factor that plays crucial roles in embryonic development and tumor progression. In many types of cancer, VEGF is overexpressed and is generally associated with tumor progression and survival rate. The polypurine/polypyrimidine sequence located upstream of the promoter region in the human VEGF gene can form specific parallel G-quadruplex structures, raising the possibility for transcriptional control of VEGF through G-quadruplex ligands.PCR stop assay, circular dichroism (CD) spectra, RNA extraction and RT-PCR, enzyme-linked immunosorbent assay (ELISA), luciferase Assays, cell scrape test, xCELLigence real-time cell analysis (RTCA), and chick embryo chorioallantoic membrane (CAM) assay.We found that quindoline derivatives can interact with the G-rich DNA sequences of the VEGF promoter to stabilize this G-quadruplex and suppress the transcription and expression of the VEGF protein. We also demonstrated that these derivatives exhibit potential anti-angiogenic activity in chick embryos and antitumor activity, including the inhibition of cell proliferation and migration.Our new findings have significances not only for understanding the mechanism of the G-quadruplex ligands mediating the VEGF transcription inhibition, but also for exploring a new anti-tumor strategy to blocking the transcription of VEGF to inhibit the angiogenesis in cancer cells.
Keywords: G-quadruplex; VEGF; Angiogenesis; Quindoline derivative; Anti-tumor;
Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5β1 or αvβ3/β5 integrin antagonists in U87MG glioma cells by Anne-Marie Ray; Florence Schaffner; Hana Janouskova; Fanny Noulet; Didier Rognan; Isabelle Lelong-Rebel; Laurence Choulier; Anne-Florence Blandin; Maxime Lehmann; Sophie Martin; Tobias Kapp; Stefanie Neubauer; Florian Rechenmacher; Horst Kessler; Monique Dontenwill (2978-2987).
Integrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5β1 and αvβ3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5β1 and αvβ3/β5 purified integrins.Small antagonists were either selective for α5β1 integrin, for αvβ3/β5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5β1 integrin in the biological assays.U87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5β1 or αvβ3/β5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5β1 integrin and was inhibited selectively by α5β1 integrin antagonists but increased by αvβ3/β5 integrin antagonists.We provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions.Our data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5β1 integrin-dependent cell migration.
Keywords: α5β1 integrin; αvβ3/β5 integrin; Small antagonist; Cell adhesion; Cell migration;