Amino Acids (v.46, #12)
Leucine stimulates ASCT2 amino acid transporter expression in porcine jejunal epithelial cell line (IPEC-J2) through PI3K/Akt/mTOR and ERK signaling pathways by Shihai Zhang; Man Ren; Xiangfang Zeng; Pingli He; Xi Ma; Shiyan Qiao (2633-2642).
Leucine has been shown to influence intestinal protein metabolism, cell proliferation and migration. Furthermore, our previous study demonstrated that branched-chain amino acids could modulate the intestinal amino acid and peptide transporters in vivo. As the possible mechanisms are still largely unknown, in the present work, we studied the transcriptional and translational regulation of leucine on amino acid transporter production in IPEC-J2 cells and the signaling pathways involved. Treatment of IPEC-J2 cells with 7.5 mM leucine enhanced the mRNA expression of the Na+-neutral AA exchanger 2 (ASCT2) and 4F2 heavy chain (4F2hc) and caused an increase in ASCT2 protein expression. Leucine also activated phosphorylation of 4E-BP1 and eIF4E through the phosphorylation of mTOR, Akt and ERK signaling pathways in IPEC-J2 cells. Pre-treatment of IPEC-J2 cells with inhibitors of mTOR and Akt (rapamycin and wortmannin) or an inhibitor of ERK (PD098059) for 30 min before leucine treatment attenuated the positive effect of leucine in enhancing the protein abundance of ASCT2. These results demonstrate that leucine could up-regulate the expression of the amino acid transporters (ASCT2) through transcriptional and translational regulation by ERK and PI3K/Akt/mTOR activation.
Keywords: Leucine; Na+-neutral AA exchanger 2; IPEC-J2 cells; ERK and PI3K/Akt/mTOR signaling pathway
Arginine protects muscle cells from wasting in vitro in an mTORC1-dependent and NO-independent manner by Daniel J. Ham; Marissa K. Caldow; Gordon S. Lynch; René Koopman (2643-2652).
Amino acids are potent regulators of muscle protein synthesis and breakdown and have received considerable attention for the treatment of muscle wasting conditions. Arginine is critically involved in numerous physiological functions including providing substrate for the production of creatine, urea and nitric oxide (NO) and in the synthesis of new proteins. However, little is known about the direct effects of arginine on skeletal muscle protein synthesis during catabolic conditions. The aims of this study were to determine whether exogenous arginine could protect skeletal muscle cells from wasting directly and whether this effect was dependent on production of NO and/or activation of the rapamycin-sensitive mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. To explore these aims, we deprived mature C2C12 myotubes from nutrients and growth factors by incubating them in HEPES buffered saline with arginine or equimolar concentrations of alanine (control). Our results show that arginine: increased the ratio of phosphorylated to total mTOR (146 %), S6 (40 %) and 4EBP1 (69 %); increased protein synthesis (69 %) during the first hour of treatment; and increased myotube diameter by ~15 %. Experiments using the NO synthase inhibitor l-NG-Nitroarginine Methyl Ester showed a NO-independent protection from muscle wasting. On the other hand, the mTORC1 inhibitor rapamycin prevented increases in phosphorylated S6, protein synthesis and myotube diameter. The activation of mTORC1 and protein synthesis by arginine was not associated with changes in the phosphorylation status of Akt, but rather increased the expression of the amino acid-sensitive type III PI3-kinase Vps34 signalling protein. These data support a direct role for arginine in the regulation of mTORC1 in skeletal muscle.
Keywords: Atrophy; Protein synthesis; C2C12; Starvation
C-type natriuretic peptide attenuates LPS-induced endothelial activation: involvement of p38, Akt, and NF-κB pathways by Gan Chen; Jingxiang Zhao; Yujing Yin; Bo Wang; Qingjun Liu; Penglong Li; Lian Zhao; Hong Zhou (2653-2663).
Endothelial activation elicited by inflammatory agents is regarded as a key event in the pathogenesis of several vascular inflammatory diseases. In the present study, the inhibitory effects and underlying mechanism of C-type natriuretic peptide (CNP) on LPS-induced endothelial activation were examined in human umbilical vein endothelial cells (HUVECs). The effect of CNP on adhesion molecule expression was assessed using quantitative real-time RT-PCR and western blotting analyses. The nuclear factor-κB (NF-κB), MAPK, and PI3K/Akt signaling pathways in LPS-stimulated HUVECs were investigated using western blotting analyses, and the production of intracellular reactive oxygen species (ROS) was measured using a fluorescence method. Pretreatment with CNP inhibited LPS-induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and P-selectin in a concentration-dependent manner. CNP suppressed the phosphorylation of p65 and NF-κB activation in LPS-stimulated cells. Moreover, CNP reduced ERK1/2 and p38 phosphorylation induced by LPS but not JNK. Furthermore, CNP induced Akt phosphorylation and activation of hemeoxygenase-1 (HO-1) expression. CNP significantly inhibited the production of intracellular ROS. These results suggest that CNP effectively attenuated LPS-induced endothelial activation by inhibiting the NF-κB and p38 signaling pathways, eliminating LPS-induced intracellular ROS production, and activating the PI3K/Akt/HO-1 pathway in HUVECs; thereby, demonstrating that CNP may be a potential therapeutic target for the treatment of sepsis and inflammatory vascular diseases.
Keywords: C-type natriuretic peptide; Endothelial activation; Lipopolysaccharide; Adhesion molecules; Nuclear factor-κB (NF-κB); Mitogen-activated protein kinase (MAPK); Hemeoxygenase-1
Improved prediction of residue flexibility by embedding optimized amino acid grouping into RSA-based linear models by Hua Zhang; Lukasz Kurgan (2665-2680).
Knowledge of protein flexibility is vital for deciphering the corresponding functional mechanisms. This knowledge would help, for instance, in improving computational drug design and refinement in homology-based modeling. We propose a new predictor of the residue flexibility, which is expressed by B-factors, from protein chains that use local (in the chain) predicted (or native) relative solvent accessibility (RSA) and custom-derived amino acid (AA) alphabets. Our predictor is implemented as a two-stage linear regression model that uses RSA-based space in a local sequence window in the first stage and a reduced AA pair-based space in the second stage as the inputs. This method is easy to comprehend explicit linear form in both stages. Particle swarm optimization was used to find an optimal reduced AA alphabet to simplify the input space and improve the prediction performance. The average correlation coefficients between the native and predicted B-factors measured on a large benchmark dataset are improved from 0.65 to 0.67 when using the native RSA values and from 0.55 to 0.57 when using the predicted RSA values. Blind tests that were performed on two independent datasets show consistent improvements in the average correlation coefficients by a modest value of 0.02 for both native and predicted RSA-based predictions.
Keywords: Residue flexibility; B-factor; Relative solvent accessibility; Linear regression; Particle swarm optimization; Amino acid grouping
Metabolomic analysis of amino acid and fat metabolism in rats with l-tryptophan supplementation by Zheng Ruan; Yuhui Yang; Yanmei Wen; Yan Zhou; Xiaofang Fu; Sheng Ding; Gang Liu; Kang Yao; Xin Wu; Zeyuan Deng; Guoyao Wu; Yulong Yin (2681-2691).
Tryptophan (TRP) is an important precursor for several neurotransmitters and metabolic regulators, which play a vital role in regulating nutrient metabolism. The purpose of this study was to investigate the effects of tryptophan supplementation on the biochemical profiles, intestinal structure, liver structure and serum metabolome in rats. Rats received daily intragastric administration of either tryptophan at doses of 200 mg/kg body weight per day or saline (control group) for 7 days. TRP supplementation had a tendency to decrease the body weight of rats (P > 0.05). The levels of urea and CHO in serum were decreased in the TRP-supplemented group rats compared with control group rats (P < 0.05). TRP supplementation increased the villus height and the ratio of villus height to crypt depth in the jejunum compared to control group rats (P < 0.05). Metabolic effects of tryptophan supplementation include: (1) increases in the serum concentrations of lysine, glycine, alanine, glutamate, glutamine, citrulline, methionine, tyrosine, 1-methylhistidine, and albumin, and decreases in the concentrations of serum branched-chain amino acid (isoleucine, valine and leucine); (2) decreases in the serum concentrations of formate and nitrogenous products (trimethylamine, TMAO, methylamine and dimethylamine), and in the contraction of trimethylamine in feces; (3) decreases in serum levels of lipids, low density lipoprotein, very low density lipoprotein, together with the elevated ratio of acetoacetate to β-hydroxybutyrate. The results indicate that tryptophan supplementation reduced the catabolism of dietary amino acids and promoted protein synthesis in rats, promoted the oxidation of fatty acid and reduced fat deposition in the body of rats.
Keywords: Tryptophan; Animo acids; Metabolism; Metabolomic; Nuclear magnetic resonance spectroscopy
The regulatory peptide apelin: a novel inhibitor of renal interstitial fibrosis by Li-Yan Wang; Zong-Li Diao; Dong-Liang Zhang; Jun-Fang Zheng; Qi-Dong Zhang; Jia-Xiang Ding; Wen-Hu Liu (2693-2704).
Epithelial–mesenchymal transition (EMT) of tubular epithelial cells is a key event in renal interstitial fibrosis and the progression of chronic kidney disease (CKD). Apelin is a regulatory peptide involved in the regulation of normal renal hemodynamics and tubular functions, but its role in renal fibrosis remains unknown. In this study, we examined the inhibitory effects of apelin on transforming growth factor-β1 (TGF-β1)-induced EMT in HK-2 cells, and evaluated its therapeutic efficacy in mice with complete unilateral ureteral obstruction (UUO). In vitro, apelin inhibited TGF-β1-mediated upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin. Increased levels of phosphorylated Smad-2/3 and decreased levels of Smad7 in TGF-β1-stimulated cells were reversed by apelin co-treatment. In the UUO model, administration of apelin significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin and laminin expression, and markedly suppressed expression of α-SMA, TGF-β1 and its type I receptor, as well as interstitial matrix components. Interestingly, in UUO mice, there was a reduction in the plasma level of apelin, which was compensated by upregulation of APJ expression in the injured kidney. Exogenous supplementation of apelin normalized the level of plasmatic apelin and renal APJ. In conclusion, our study provides the first evidence that apelin is able to ameliorate renal interstitial fibrosis by suppression of tubular EMT through a Smad-dependent mechanism. The apelinergic system itself may promote some compensatory response in the renal fibrotic process. These results suggest that apelin has potential renoprotective effects and may be an effective agent for retarding CKD progression.
Keywords: Apelin; Chronic kidney disease; Fibrosis; Tubule cells
BP8, a novel peptide from avian immune system, modulates B cell developments by Xiao-Dong Liu; Bin Zhou; Xiu-Li Feng; Rui-Bing Cao; Pu-Yan Chen (2705-2713).
The bursa of Fabricius (BF) is the key humoral immune organ unique to birds, and is critical for early B-lymphocyte proliferation and differentiation. However, the molecular basis and mechanisms through which the BF regulates B cell development are not fully understood. In this study, we isolated and identified a new bursal peptide (BP8, AGHTKKAP) by RP-HPLC and MALDI-TOF–MS. BP8 promoted colony-forming pre-B formation, bound B cell precursor, regulated B cell development in vitro as well as in vivo, upstream of the EBF-E2A-Pax5 regulatory complex and increased immunoglobulin secretion. These data revealed a bursal-derived multifunctional factor BP8 as a novel biomaterial which is essential for the development of the immune system. This study elucidates further the mechanisms involved in humoral immune system and has implications in treating human diseases.
Keywords: Bursal peptide (BP8); Immunomodulatory function; B cell development
Synthesis and in vitro evaluation of thiododecaborated α, α- cycloalkylamino acids for the treatment of malignant brain tumors by boron neutron capture therapy by Yoshihide Hattori; Shintaro Kusaka; Mari Mukumoto; Miki Ishimura; Yoichiro Ohta; Hiroshi Takenaka; Kouki Uehara; Tomoyuki Asano; Minoru Suzuki; Shin-ichiro Masunaga; Koji Ono; Shinji Tanimori; Mitsunori Kirihata (2715-2720).
Boron-neutron capture therapy (BNCT) is an attractive technique for cancer treatment. As such, α, α-cycloalkyl amino acids containing thiododecaborate ([B12H11]2−-S-) units were designed and synthesized as novel boron delivery agents for BNCT. In the present study, new thiododecaborate α, α-cycloalkyl amino acids were synthesized, and biological evaluation of the boron compounds as boron carrier for BNCT was carried out.
Keywords: Boron neutron capture therapy; Boron cluster; Boronated amino acid; Immunocytochemistry
In vivo NIRF imaging-guided delivery of a novel NGR–VEGI fusion protein for targeting tumor vasculature by Wenhui Ma; Guoquan Li; Jing Wang; Weidong Yang; Yingqi Zhang; Peter S. Conti; Kai Chen (2721-2732).
Pathological angiogenesis is crucial in tumor growth, invasion and metastasis. Previous studies demonstrated that the vascular endothelial growth inhibitor (VEGI), a member of the tumor necrosis factor superfamily, can be used as a potent endogenous inhibitor of tumor angiogenesis. Molecular probes containing the asparagine–glycine–arginine (NGR) sequence can specifically bind to CD13 receptor which is overexpressed on neovasculature and several tumor cells. Near-infrared fluorescence (NIRF) optical imaging for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The aim of this study was to develop a new NIRF imaging probe on the basis of an NGR–VEGI protein for the visualization of tumor vasculature. The NGR–VEGI fusion protein was prepared from prokaryotic expression, and its function was characterized in vitro. The NGR–VEGI protein was then labeled with a Cy5.5 fluorophore to afford Cy5.5-NGR–VEGI probe. Using the NIRF imaging technique, we visualized and quantified the specific delivery of Cy5.5-NGR–VEGI protein to subcutaneous HT-1080 fibrosarcoma tumors in mouse xenografts. The Cy5.5-NGR–VEGI probe exhibited rapid HT-1080 tumor targeting, and highest tumor-to-background contrast at 8 h post-injection (pi). Tumor specificity of Cy5.5-NGR–VEGI was confirmed by effective blocking of tumor uptake in the presence of unlabeled NGR–VEGI (20 mg/kg). Ex vivo NIRF imaging further confirmed in vivo imaging findings, demonstrating that Cy5.5-NGR–VEGI displayed an excellent tumor-to-muscle ratio (18.93 ± 2.88) at 8 h pi for the non-blocking group and significantly reduced ratio (4.92 ± 0.75) for the blocking group. In conclusion, Cy5.5-NGR–VEGI provided highly sensitive, target-specific, and longitudinal imaging of HT-1080 tumors. As a novel theranostic protein, Cy5.5-NGR–VEGI has the potential to improve cancer treatment by targeting tumor vasculature.
Keywords: NIRF imaging; Tumor vasculature; CD13 receptor; NGR; Vascular endothelial growth inhibitor
Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin by Vivian Asante; Jérémie Mortier; Gerhard Wolber; Beate Koksch (2733-2744).
Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1′or P2′ positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC 188.8.131.52) or the aspartic protease, pepsin (EC 184.108.40.206). The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure–activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.
Keywords: Chymotrypsin; Pepsin; Aminobutyric acid; Difluoroethylglycine; Trifluoroethylglycine
Novel fluorinated lipopeptides from Bacillus sp. CS93 via precursor-directed biosynthesis by Neil K. O’Connor; Alex S. Hudson; Steven L. Cobb; Deborah O’Neil; Jennifer Robertson; Vanessa Duncan; Cormac D. Murphy (2745-2752).
While attempting to improve production of fluoro-iturin A in Bacillus sp. CS93 new mono- and di-fluorinated fengycins were detected in culture supernatants by 19F NMR and tandem mass spectrometry, after incubation of the bacterium with 3-fluoro-l-tyrosine. The fluorinated amino acid was presumably incorporated in place of one or both of the tyrosyl residues in fengycin. Investigations to generate additional new fluorinated derivatives were undertaken using commercially available fluorinated phenylalanines and 2-fluoro- and 2,3-difluoro-tyrosine that were synthesised by Negishi cross-coupling of iodoalanine and fluorinated bromo-phenols. The anti-fungal activity of the fluorinated lipopeptides was assayed against Trichophyton rubrum and found to be similar to that of the non-fluorinated metabolites.
Keywords: Antibiotic; Bacillus; Fluorine; Tyrosine
β,γ-Diamino acid: an original building block for hybrid α/γ-peptide synthesis with extra hydrogen bond donating group by Andrii Stanovych; Régis Guillot; Cyrille Kouklovsky; Emeric Miclet; Valérie Alezra (2753-2757).
Using a β,γ-diamino acid, several small hybrid α/γ peptides have been synthesized and their conformations investigated through extensive NMR studies and molecular dynamics. A tripeptide and a tetrapeptide have thus shown several hydrogen bonds in solution, including a 13-membered ring involving the β-nitrogen.
Keywords: Peptide; γ-amino acid; Foldamer; NMR; Molecular dynamics; Structure
Melittin peptides exhibit different activity on different cells and model membranes by Elaheh Jamasbi; Steven Batinovic; Robyn A. Sharples; Marc-Antoine Sani; Roy Michael Robins-Browne; John D. Wade; Frances Separovic; Mohammed Akhter Hossain (2759-2766).
Melittin (MLT) is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogs were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes and hemolysis. Although lytic activity against model membranes decreased for the MLT dimer, hemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation.
Keywords: Melittin; Antimicrobial peptide; Cytotoxicity; Hemolysis; Dye leakage
The spermidine analogue GC7 (N1-guanyl-1,7-diamineoheptane) induces autophagy through a mechanism not involving the hypusination of eIF5A by Serafina Oliverio; Marco Corazzari; Claudia Sestito; Lucia Piredda; Giuseppe Ippolito; Mauro Piacentini (2767-2776).
The exogenous administration of spermidine promotes longevity in many model organisms. It has been proposed that this anti-age activity of spermidine is related to this polyamine’s ability to promote autophagy. Since spermidine is the substrate for the eIF5A post-translational modification by hypusination, we asked ourselves whether mature eIF5A may represent the link between spermidine and autophagy induction. To test this hypothesis, we inhibited the conversion of native eIF5A by a pharmacological approach, using the N1-guanyl-1,7-diamineoheptane (GC7), a spermidine analogue which competitively and reversibly inhibits deoxyhypusine synthase (DHS). In addition, we also employed genetic approaches by ablating both the eIF5A protein itself and DHS, the rate limiting enzyme catalyzing the conversion of lysine to hypusine. Collectively the data presented in this study demonstrate that the mature eIF5A (hypusinated form) is not involved in the autophagic pathway and that the inhibitor of DHS, GC7, produces off-target effect(s) resulting in marked induction of basal autophagy. These data are relevant in light of the fact that GC7 is considered a potent and selective inhibitor of DHS and is a potential candidate drug for cancer, diabetes and HIV therapy.
Keywords: eIF5A; Autophagy; GC7; DHS; Cancer; HIV
Antioxidant activity of caffeoyl-prolyl-histidine amide and its effects on PDGF-induced proliferation of vascular smooth muscle cells by Seon-Yeong Kwak; Hyun Jung Lee; Jin-Kyoung Yang; Eun Jig Lee; MiRan Seo; Yoon-Sik Lee (2777-2785).
Caffeic acid (CA) is one of the antioxidants found in plants, which protects vascular cells against vascular injuries from oxidative stress. In our previous study, caffeoyl-prolyl-histidine amide (CA-L-Pro-L-His-NH2; CA-PH; a CA derivative) was synthesized, which exhibited a strong antioxidant activity with sufficient stability. In this study, we investigated the role of CA-PH in vascular smooth muscle cells (VSMCs) and confirmed the enhanced antioxidant activity of CA-PH compared with that of CA. In in vitro tube assays, CA-PH showed a higher free-radical-scavenging activity and lipid-peroxidation-inhibition activity than those of CA. In VSMCs, CA-PH significantly reduced hydrogen peroxide-induced ROS generation and increased the expression of heme oxygenase-1. Moreover, CA-PH effectively inhibited the platelet-derived growth factor-induced cellular proliferation of VSMCs, which was confirmed by a decrease in the expression of the proliferating cell nuclear antigen and the phosphorylation of Akt.
Keywords: Caffeoyl-prolyl-histidine amide; Antioxidant; Vascular smooth muscle cells; Heme oxygenase-1
Sepsis-induced changes in amino acid transporters and leucine signaling via mTOR in skeletal muscle by Lacee J. Laufenberg; Anne M. Pruznak; Maithili Navaratnarajah; Charles H. Lang (2787-2798).
The present study tested the hypothesis that sepsis-induced leucine (Leu) resistance in skeletal muscle is associated with a down-regulation of amino acid transporters important in regulating Leu flux or an impairment in the formation of the Leu-sensitive mTOR–Ragulator complex. Sepsis in adult male rats decreased basal protein synthesis in gastrocnemius, associated with a reduction in mTOR activation as indicated by decreased 4E-BP1 and S6K1 phosphorylation. The ability of oral Leu to increase protein synthesis and mTOR kinase after 1 h was largely prevented in sepsis. Sepsis increased CAT1, LAT2 and SNAT2 mRNA content two- to fourfold, but only the protein content for CAT1 (20 % decrease) differed significantly. Conversely, sepsis decreased the proton-assisted amino acid transporter (PAT)-2 mRNA by 60 %, but without a coordinate change in PAT2 protein. There was no sepsis or Leu effect on the protein content for RagA-D, LAMTOR-1 and -2, raptor, Rheb or mTOR in muscle. The binding of mTOR, PRAS40 and RagC to raptor did not differ for control and septic muscle in the basal condition; however, the Leu-induced decrease in PRAS40·raptor and increase in RagC·raptor seen in control muscle was absent in sepsis. The intracellular Leu concentration was increased in septic muscle, compared to basal control conditions, and oral Leu further increased the intracellular Leu concentration similarly in both control and septic rats. Hence, while alterations in select amino acid transporters are not associated with development of sepsis-induced Leu resistance, the Leu-stimulated binding of raptor with RagC and the recruitment of mTOR/raptor to the endosome-lysosomal compartment may partially explain the inability of Leu to fully activate mTOR and muscle protein synthesis.
Keywords: Sepsis; Protein synthesis; mTOR; Amino acid transporters; Rag GTPases; PRAS40; Ragulator
Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization by Jörg Ziegler; Steffen Abel (2799-2808).
A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).
Keywords: Arabidopsis thaliana ; Amino acids; Mass spectrometry; Derivatization; Root meristems
Metformin inhibits the invasion of human hepatocellular carcinoma cells and enhances the chemosensitivity to sorafenib through a downregulation of the ERK/JNK-mediated NF-κB-dependent pathway that reduces uPA and MMP-9 expression by Shu-Ching Hsieh; Jen-Pi Tsai; Shun-Fa Yang; Meng-Ju Tang; Yi-Hsien Hsieh (2809-2822).
Metformin has been shown to exert anti-cancer activities in several cancer cells and animal models. However, the molecular mechanisms of its anti-metastatic activities remain poorly understood and warrant further investigation. The aims of this study were to evaluate the ability of metformin to inhibit the migration and invasion of hepatocellular carcinoma (HCC) cells and identify its effects on signaling pathways. Our data indicate that metformin inhibits the migration and invasion of human HCC cells. Metformin was also found to significantly inhibit the expression and secretion of MMP-9 and uPA in HCC cells, and suppress the phosphorylation of ERK1/2 and JNK1/2. Treatment with an ERK1/2 inhibitor (PD98059) or JNK1/2 inhibitor (SP600125) enhanced the inhibitory effects of metformin on the migration and invasion of HCC cells. Moreover, metformin-induced inhibition of MMP-9 and uPA promoter activity also blocked the nuclear translocation of NF-κB and its binding to the MMP-9 and uPA promoters, and these suppressive effects were further enhanced by PD98059 or SP600125. Moreover, metformin markedly enhanced the anti-metastatic effects of sorafenib. In conclusion, metformin inhibits the migration and invasion of HCC cells by suppressing the ERK/JNK-mediated NF-κB-dependent pathway, and thereby reducing uPA and MMP-9 expression. Additionally, combination treatment with metformin and sorafenib yielded synergistic inhibitory effects in suppressing cell migration and invasion of HCC cells. These findings provide insight into the molecular mechanisms involved in the anti-metastatic effects of metformin, as well as its ability to enhance the chemosensitivity of HCC cells to sorafenib.
Keywords: Hepatocellular carcinoma cells; Metformin; Invasion; Migration; MMP-9; uPA; Sorafenib
Easy preparation of dehydroalanine building blocks equipped with oxazolidin-2-one chiral auxiliaries, and applications to the stereoselective synthesis of substituted tryptophans by Rossella De Marco; Lorenzo Cavina; Arianna Greco; Luca Gentilucci (2823-2839).
Chiral dehydroamino acid building blocks are versatile starting materials for the preparation of optically active unusual amino acids and other compounds of pharmacological interest. Herein we disclose the expedient preparation of dehydroalanines (ΔAla) equipped with oxazolidin-2-one (Oxd) chiral auxiliaries, Ts-Oxd-ΔAla-OMe. These compounds have been obtained in high yields from dipeptides Ts-Ser/Thr/phenylSer-Ser-OMe by the one-pot cyclization–elimination reaction with N,N-disuccinimidyl carbonate and catalytic DIPEA. To test the efficacy of the chiral auxiliaries in controlling asymmetric transformations, the Friedel–Crafts alkylations of indoles carrying diverse substituents were performed in the presence of Lewis and Brønsted acids. The reactions proceeded with good to excellent diastereomeric ratios giving (S)- or (R)-tryptophan derivatives, isolated very conveniently by simple flash chromatography. To verify the utility of this approach, optically pure (S)-2-methyltryptophan and (S)-5-fluorotryptophan were obtained and utilized to prepare analogues of endogenous opioid peptide endomorphin-1, H-Tyr-Pro-Trp-PheNH2.
Keywords: Dehydroamino acids; Oxazolidinone chiral auxiliary; Tryptophan; Friedel–Crafts alkylation; Endomorphin-1
Structure of the cyclic peptide [W8S]contryphan Vn: effect of the tryptophan/serine substitution on trans–cis proline isomerization by Ridvan Nepravishta; Walter Mandaliti; Sonia Melino; Tommaso Eliseo; Maurizio Paci (2841-2853).
The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S–S bridge between two cysteines and a d-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans–cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans–cis isomerization, this being also an important step in protein folding.
Keywords: Contryphan; NMR; Cyclic peptides; Three-dimensional structure; Fluorescence; Proline isomerization