Marine Drugs

3162 KiB

Open AccessArticle

Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation

by Ikuko Ito, Toshikazu Yoneda, Yoshihiko Omura, Tomohiro Osaki, Shinsuke Ifuku, Hiroyuki Saimoto, Kazuo Azuma, Tomohiro Imagawa, Takeshi Tsuka, Yusuke Murahata, Norihiko Ito, Yoshiharu Okamoto and Saburo Minami

Mar. Drugs 2015, 13(12), 7463-7475; https://doi.org/10.3390/md13127076 - 19 Dec 2015

Cited by 13 | Viewed by 5941

Abstract

Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils [...] Read more.

Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm²), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation. Full article

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1433 KiB

Open AccessArticle

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis

by Claire Bouvard, Isabelle Galy-Fauroux, Françoise Grelac, Wassila Carpentier, Anna Lokajczyk, Sophie Gandrille, Sylvia Colliec-Jouault, Anne-Marie Fischer and Dominique Helley

Mar. Drugs 2015, 13(12), 7446-7462; https://doi.org/10.3390/md13127075 - 18 Dec 2015

Cited by 31 | Viewed by 6715

Abstract

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein [...] Read more.

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF’s mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair. Full article

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Bio-Plex analysis of signal transduction induced by LMWF, in association with FGF-2 and VEGF on ECFCs (a) and HUVECs (b). Phosphorylation of AKT, ERK, p38 and JNK in response to the different treatments: no treatment (ctrl); LMWF (10 μg/mL), FGF-2 (5 ng/mL); FGF-2 (5 ng/mL) and LMWF (10 μg/mL); VEGF (40 ng/mL); VEGF (40 ng/mL) and LMWF (10 μg/mL). Data are ratios of phospho-protein to the total protein, normalized to the untreated group (ctrl) and are represented as the mean + SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated group (ctrl). # p < 0.05, ## p < 0.01 vs. VEGF treated group. Full article ">Figure 2
LMWF but not LMWH induces the phosphorylation of AKT in ECFCs (a) and HUVECs (b) in a time dependent manner. Cells were treated with LMWF or with LMWH (10 μg/mL) for 5, 10, 15, 45 and 120 min and then washed and lysed. Phosphorylated AKT and total AKT were quantified by Western blot analysis. Data are represented as a ratio of pAKT to AKT and are the mean ± SEM of at least 3 independent experiments. * p < 0.05, ** p < 0.01 vs. LMWH treated group. Full article ">Figure 3
LMWF but not LMWH induces cell migration in a PI3K-dependent manner. ECFC (a) or HUVEC (b) monolayers were mechanically scratched with a sterile plastic pipette tip after a 24 h incubation with the following treatments: no treatment (ctrl); LMWF (10 μg/mL); LMWH (10 μg/mL); wortmannin (W, 100 nM); wortmannin (W, 100 nM) and LMWF (10 μg/mL); wortmannin (W, 100 nM) and LMWH (10 μg/mL). Microphotographs were taken just after the scratch and 6 h later to quantify the surface covered by migrating cells (between the solid and the dash-dot lines). Data are normalized to the untreated group and are the mean + SEM of 3 independent experiments. ** p < 0.01, *** p < 0.001 vs. all other groups. Full article ">Figure 4
Gene expression heatmaps generated using illumina beadchips data. ECFCs (a,b) and HUVECs (c,d) were treated with 10 μg/mL of LMWF for 6 h (a,c) or 24 h (b,d). RNA was extracted and analyzed using Illumina Human HT-12 Expression beadchips. The heatmaps show the expression fold change of a selection of differentially expressed genes, with an expression fold change greater than 1.5 fold and a significance p-value < 0.05. An up-regulation will appear red and a down-regulation will appear green. At least four biologically independent replicates are represented. Full article ">Figure 5
Gene expression heatmap generated using qRT-PCR data. ECFCs and HUVECs were treated with 10 μg/mL of LMWF for 6 h or 24 h. RNA was extracted, cDNA was synthesized and analyzed using Taqman technology. mRNA levels were measured by quantitative RT-PCR using total RNA isolated from ECFC or HUVEC. The heatmap shows for each gene the mean of the expression fold change obtained from at least four biologically independents replicates. An up-regulation will appear red and a down-regulation will appear green. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. untreated control. Full article ">

2175 KiB

Open AccessArticle

Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX

by Chao Lin, Zeng-Shan Liu, Dong-Xu Wang, Lin Li, Pan Hu, Sheng Gong, Yan-Song Li, Cheng Cui, Zong-Cheng Wu, Yang Gao, Yu Zhou, Hong-Lin Ren and Shi-Ying Lu

Mar. Drugs 2015, 13(12), 7433-7445; https://doi.org/10.3390/md13127066 - 17 Dec 2015

Cited by 15 | Viewed by 7961

Abstract

Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to [...] Read more.

Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb). Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX) strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA) analysis, four candidate aptamers (O24, O31, O39, O40) were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 10⁸ M⁻¹, 1.47 × 10⁹ M⁻¹, 1.23 × 10⁹ M⁻¹ and 1.05 × 10⁹ M⁻¹, respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X − 1.78. The IC₅₀ of O31 was 3.39 ng·mL⁻¹, which was close to the value predicted by the OA ELISA (IC₅₀ = 4.4 ng·mL⁻¹); the IC₁₀ was 0.33 ng·mL⁻¹. The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay. Full article

(This article belongs to the Special Issue Okadaic Acid and Dinophysis Toxins)

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SELEX and Reaction Scheme. (A) The SELEX scheme via magnetic beads. First, the F(ab′)2 fragment was incubated with tosylactivated dynabeads in binding buffer. To ensure high binding efficiency, the optimal concentration used for the protein and dynabeads was 3 μg F(ab′)2 fragment/107 dynabeads. Second, the ssDNA library was added to the pre-coated dynabeads for incubation with the target protein. To ensure highly stringent binding conditions, the ratio of ssDNA and target protein was 1:200. Third, the superparamagnetic beads carrying the target protein and target-bound aptamers were trapped using a magnetic rack. The unbound ssDNA was removed via stringent washing. The bound aptamers were eluted from the beads by heating to 80 °C. The positive aptamers were isolated via magnetic separation. Fourth, the product of selected aptamers was amplified by PCR with a 5′-phosphorylated reverse primer. Next, single-stranded DNA was generated by adding the lambda exonuclease that selectively digests the 5′ phosphorylated strand of the dsDNA. The above four steps were repeated for 10-12 rounds. Finally, the isolated aptamers were cloned and sequenced to obtain candidate aptamers. (B,C) Two alternative assays for the detection of OA based on aptamers; In (B), the biotin-aptamer, as a substitute of OA standard, could limit the binding between the OA-mAb pre-coated and OA toxin in samples; In (C), the aptamer, as a substitute of OA standard, could limit the binding between the OA-mAb and the OA-BSA pre-coated in the carrier. Full article ">Figure 2
Transmission electron microscopy of the superparamagnetic beads. (A) Transmission electron microscopy of tosylactivated dynabeads. In the scale of the TEM, the magnetic beads were uniformly dispersed and possessed of a consistent size; (B) Transmission electron microscopy of beads coated with F(ab′)2 fragments. The superparamagnetic beads carrying the target protein were verified by transmission electron microscopy. In (B), a “protein cloud” can be observed around the superparamagnetic beads. The F(ab′)2 fragment was stained with phosphotungstic acid; thus, the protein on the face of the magnetic beads was dark. Full article ">Figure 3
The rounds of SELEX. A biotin-avidin ELISA was used to test the OD values of rounds of selection using the ssDNA pool. The OD value of each round was expressed as the mean ± SD (n = 3). Counter selection was performed using other related antibody-coated magnetic beads after rounds five and seven. After the 12th round of selection, the ssDNA library enrichment had reached saturation. Full article ">Figure 4
(A) The affinity of random clones. The OD values of the forty cloned sequences were detected using indirect ELISA. O24, O31, O39 and O40 were chosen as candidate aptamers because of their higher affinity; (B) Predicted secondary structures of four candidate aptamers. Predicted secondary structures of aptamers O24, O31, O39 and O40 determined by the mfold tool. Full article ">Figure 5
Characterization of the O31 aptamer. (A) The specificity of the O31 aptamer. The six monoclonal antibodies against the marine toxin (as prepared previously by our lab [<a href="#B32-marinedrugs-13-07066" class="html-bibr">32</a>,<a href="#B33-marinedrugs-13-07066" class="html-bibr">33</a>,<a href="#B34-marinedrugs-13-07066" class="html-bibr">34</a>,<a href="#B35-marinedrugs-13-07066" class="html-bibr">35</a>]), were used to test the specificity of O31. The OA-mAb and six other monoclonal antibodies were coated on magnetic beads. Then, the proper dilution of the O31 aptamer was added to the tubes. The OD492 values were measured to analyse the specificity; (B) Affinity constants value for the O31 aptamer. The synthetic biotin-aptamers were added at concentrations of 0.1, 0.155, 0.31, 0.625, 1.25, 2.5 and 5 ng·mL−1 to 1, 2, 4, and 8 μg·mL−1 of OA-mAb pre-coated on the plate. Full article ">Figure 6
(A) The calibration curves for OA by direct competitive ELISA (dc-ELISA). The calibration curves for OA were developed using different dilutions of free OA toxin. The regression equation was Y = 1.312 − 0.686X; (B) The calibration curves for O31 by dc-ELISA. The calibration curves for O31 were developed using different dilutions of biosynthetic aptamer. The regression equation was Y = 3.092 − 4.099X. The relationship equation between OA and O31 was Y = 2.185X − 1.78, where Y represents the aptamer content and X represents the OA content; (C) The determination of the O31 IC50 value. The regression equation was Y = 29.08 + 39.41X. The 50% inhibition concentration (IC50) of O31 was 3.39 ng·mL−1 and the IC10 was 0.33 ng·mL−1. Full article ">

1500 KiB

Open AccessArticle

Structure-Activity Relationship and in Vivo Anti-Tumor Evaluations of Dictyoceratin-A and -C, Hypoxia-Selective Growth Inhibitors from Marine Sponge

by Yuji Sumii, Naoyuki Kotoku, Akinori Fukuda, Takashi Kawachi, Masayoshi Arai and Motomasa Kobayashi

Mar. Drugs 2015, 13(12), 7419-7432; https://doi.org/10.3390/md13127074 - 16 Dec 2015

Cited by 18 | Viewed by 6440

Abstract

Oral dictyoceratin-C (1) and A (2), hypoxia-selective growth inhibitors, showed potent in vivo antitumor effects in mice subcutaneously inoculated with sarcoma S180 cells. Structurally modified analogs were synthesized to assess the structure–activity relationship of the natural compounds 1 and [...] Read more.

Oral dictyoceratin-C (1) and A (2), hypoxia-selective growth inhibitors, showed potent in vivo antitumor effects in mice subcutaneously inoculated with sarcoma S180 cells. Structurally modified analogs were synthesized to assess the structure–activity relationship of the natural compounds 1 and 2 isolated from a marine sponge. Biological evaluation of these analogs showed that the exo-olefin and hydroxyl and methyl ester moieties were important for the hypoxia-selective growth inhibitory activities of 1 and 2. Thus far, only substitution of the methyl ester with propargyl amide in 1 was found to be effective for the synthesis of probe molecules for target identification. Full article

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1202 KiB

Open AccessReview

Distribution in Different Organisms of Amino Acid Oxidases with FAD or a Quinone As Cofactor and Their Role as Antimicrobial Proteins in Marine Bacteria

by Jonatan C. Campillo-Brocal, Patricia Lucas-Elío and Antonio Sanchez-Amat

Mar. Drugs 2015, 13(12), 7403-7418; https://doi.org/10.3390/md13127073 - 16 Dec 2015

Cited by 18 | Viewed by 6517

Abstract

Amino acid oxidases (AAOs) catalyze the oxidative deamination of amino acids releasing ammonium and hydrogen peroxide. Several kinds of these enzymes have been reported. Depending on the amino acid isomer used as a substrate, it is possible to differentiate between l-amino acid [...] Read more.

Amino acid oxidases (AAOs) catalyze the oxidative deamination of amino acids releasing ammonium and hydrogen peroxide. Several kinds of these enzymes have been reported. Depending on the amino acid isomer used as a substrate, it is possible to differentiate between l-amino acid oxidases and d-amino acid oxidases. Both use FAD as cofactor and oxidize the amino acid in the alpha position releasing the corresponding keto acid. Recently, a novel class of AAOs has been described that does not contain FAD as cofactor, but a quinone generated by post-translational modification of residues in the same protein. These proteins are named as LodA-like proteins, after the first member of this group described, LodA, a lysine epsilon oxidase synthesized by the marine bacterium Marinomonas mediterranea. In this review, a phylogenetic analysis of all the enzymes described with AAO activity has been performed. It is shown that it is possible to recognize different groups of these enzymes and those containing the quinone cofactor are clearly differentiated. In marine bacteria, particularly in the genus Pseudoalteromonas, most of the proteins described as antimicrobial because of their capacity to generate hydrogen peroxide belong to the group of LodA-like proteins. Full article

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Cysteine tryptophylquinone (CTQ) cofactor. Full article ">Figure 2
Phylogenetic relationships of enzymes with amino acid oxidase activity. The tree was created by the Neighbor-Joining method integrated in the program MEGA6 [<a href="#B23-marinedrugs-13-07073" class="html-bibr">23</a>]. Sequences were aligned using the program MUSCLE built in MEGA6. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. Numbers at branches indicate bootstrap values higher than 70% for both Neighbor-Joining and Maximum Likelihood trees. The colored groups are detailed in <a href="#marinedrugs-13-07073-f003" class="html-fig">Figure 3</a> and <a href="#marinedrugs-13-07073-f004" class="html-fig">Figure 4</a>. LAAOs, l-amino acid oxidases; DAAO, d-amino acid oxidases; LASPOs, l-aspartate oxidases. Full article ">Figure 3
Phylogenetic relationships of representative LodA-like proteins (A), d-amino acid oxidases (B) and l-aspartate oxidases (C); The tree was created by the Neighbor-Joining method integrated in the program MEGA6 [<a href="#B23-marinedrugs-13-07073" class="html-bibr">23</a>]. Sequences were aligned using the program MUSCLE built in MEGA6. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. Numbers at branches indicate bootstrap values higher than 70% for both Neighbor-Joining and Maximum Likelihood trees. An asterisk indicates that this branch was not detected, or it had a value lower than 70%. Full article ">Figure 4
Phylogenetic relationships of representative l-amino acid oxidases in vertebrates (A), gastropods (B) and fungi (C). The tree was created by the Neighbor-Joining method integrated in the program MEGA6 [<a href="#B23-marinedrugs-13-07073" class="html-bibr">23</a>]. Sequences were aligned using the program MUSCLE built in MEGA6. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. Numbers at branches indicate bootstrap values higher than 70% for both Neighbor-Joining and Maximum Likelihood trees. Full article ">

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Open AccessArticle

Algal Toxin Azaspiracid-1 Induces Early Neuronal Differentiation and Alters Peripherin Isoform Stoichiometry

by Linda V. Hjørnevik, Ann K. Frøyset, Toril A. Grønset, Krisna Rungruangsak-Torrissen and Kari E. Fladmark

Mar. Drugs 2015, 13(12), 7390-7402; https://doi.org/10.3390/md13127072 - 14 Dec 2015

Cited by 9 | Viewed by 5917

Abstract

Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to [...] Read more.

Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s). A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin. Full article

(This article belongs to the Special Issue Marine Neurotoxins)

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Figure 1
Concentration-dependent effect of AZA-1 on PC12 cell viability. PC12 cells were exposed to increasing dosages of AZA-1 or corresponding solvent control. Cell viability was determined by CCK-8 assay. Values are the mean ± SD (n = 2–3). Full article ">Figure 2
Effect of AZA-1 on PC12 morphology and cell viability. PC12 cells were treated with 15 nM AZA-1 for 6 h to 72 h, or negative control. (A) Cell morphology after AZA-1 exposure. AZA-treated cells appeared more differentiated with neurite-like protrusions compared to controls. Bar, 20 μm. (B) Quantitation of neurite-like protrusions. C.d. = cell diameter. (C) Cell viability as determined based on dehydrogenase activity using CCK-8 assay. Data are presented as mean ± SD (n = 9) of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.01). (D) Reduction of cell number after AZA-1 exposure. Data are shown as mean ± SD (n = 3). Full article ">Figure 3
Neurite-like protrusions are strongly stained with peripherin. AZA-1 exposed (15 nM, 24 h) PC12 cells (E-I) and controls (A-C) were processed for immuno-labelling using anti-peripherin (red). Panel D shows relative mean cell fluorescence ± sem (n = 30). Panel H/I show negative control with no primary antibody added. Nuclei were stained with DAPI (blue). Full article ">Figure 4
Time-dependent effect of AZA-1 on the neuronal differentiation markers peripherin, tubulinβ3, and tyrosine hydroxylase. Cells were exposed to 15 nM AZA-1 or negative control for up to 24 h and differentiation marker expression was analysed by Western blotting. (A) Three peripherin bands were observed whereas the second band (indicated by an asterisk) was down-regulated at all examined time points after addition of AZA-1; (B) The blot was then stripped and reprobed with anti-tubulinβ3; (C) Tyrosine hydroxylase expression. PonS stainings (A,B) and (C) are shown as loading control. Full article ">Figure 5
The down-regulated peripherin isoform is not found in the insoluble fraction. Cells were treated with 15 nM AZA-1or solvent control for 24 h and lysed in homogenisation buffer. The insoluble pellet obtained after centrifugation was dissolved in SDS-containing sample buffer. Western blot analysis with peripherin antibody showed that the second peripherin band disappeared both in the soluble and insoluble fraction after AZA-1 treatment. PonS staining was used as loading control. Molecular weight in kDa. Full article ">Figure 6
Identification of peripherin isoforms by mass spectrometry. (A) Proteins from total cell lysates (15 nM AZA-1 or negative control) were separated by SDS-PAGE. The gels were either further processed for analysis by Western blotting using a peripherin antibody or stained with Coomassie. The Western blot was used as guidance for excision of five gel slices (from the control lysate) of the Coomassie-stained gel. The proteins in the gel slices were in-gel digested and analysed by mass spectrometry. Peripherin isoforms were identified in all five gel slices (see <a href="#marinedrugs-13-07072-t001" class="html-table">Table 1</a>). (B) Structure of the rat peripherin gene (ENSRNOG00000015643; 1 September 2014). The gene consists of 9 exons, labelled 1–9. Grey areas indicate protein-coding sequences. There are two start codons in-frame with the longest open reading frame, of which the second (ATG2) is considered the canonical start codon. Normally all introns are spliced out to give rise to peripherin. Dotted black lines indicates a possible alternative splicing event in which intron 4 (I4) is retained, giving rise to a 96-bp-long insert between exon 4 and exon 5 in the mature mRNA. This splicing event has previously been shown to take place in mouse peripherin mRNA and is named Pe-61. (C) The two possible mRNAs resulting from the splicing events described in (B). Retention of I4 gives rise to a 32 amino acid long insert (underlined in black in the protein sequence). A tryptic peptide unique for the peripherin isoform CRA_a/Pe-61 which is identified by mass spectrometry is labelled in red. As can be seen, parts of intron 4 connected to exon 5 was identified with the mass spectrometer, indicating that this isoform of peripherin does exist in rats. Note that isoform CRA_a/Pe-61 uses the first in-frame start codon, resulting in 6 additional amino acids on its N-terminal and hence a different numbering of the amino acids. (D) The N-terminal amino acid sequence of peripherin and the predicted peripherin isoform CRA_b, in which the first in-frame start codon is used. Our mass spectrometry data identified a tryptic peptide containing five of the six most N-terminal amino acids in peripherin isoform CRA_b. (E) Mass spectrum of the tryptic peptide consisting of parts of intron 4 connected to exon 5 in peripherin isoform CRA_a/Pe-61. (F) Mass spectrum of the N-terminal tryptic peptide in peripherin isoform CRA_b. Full article ">Figure 7
The proteasome, caspases and calpains are not involved in peripherin down-regulation after AZA-1 exposure. Cells were pre-incubated for 1 h with a proteasome inhibitor (MG132), caspase inhibitor (Z-VAD-FMK) or calpain inhibitor (PD150606) prior to exposure to 15 nM AZA-1 or negative control for 24 h. Expression of peripherin isoforms (arrows) was detected using Western blotting. Pre-incubation with the different inhibitors did not prevent down-regulation of the second peripherin band (indicated by an asterisk). It was noted that pre-treatment with the proteasome inhibitor MG132 alone also led to a down-regulation of the second peripherin band (third lane from left). Full article ">

2854 KiB

Open AccessArticle

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

by Imtiaj Hasan, Shigeki Sugawara, Yuki Fujii, Yasuhiro Koide, Daiki Terada, Naoya Iimura, Toshiyuki Fujiwara, Keisuke G. Takahashi, Nobuhiko Kojima, Sultana Rajia, Sarkar M. A. Kawsar, Robert A. Kanaly, Hideho Uchiyama, Masahiro Hosono, Yukiko Ogawa, Hideaki Fujita, Jiharu Hamako, Taei Matsui and Yasuhiro Ozeki

Mar. Drugs 2015, 13(12), 7377-7389; https://doi.org/10.3390/md13127071 - 14 Dec 2015

Cited by 41 | Viewed by 9036

Abstract

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite [...] Read more.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface. Full article

(This article belongs to the Special Issue Marine Glycoconjugates)

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Different cell agglutination activities of MytiLec. MytiLec (0, 10, and 50 μg/mL) was applied to Ramos (5 × 105 cells) and K562 (2 × 105 cells) cells and observed by phase contrast microscopy. Full article ">Figure 2
Reduction of cell viability by MytiLec. (A) Determination of viability by WST-8 assay. Dotted columns: Ramos. White columns: K562. Cells (2 × 105 of Ramos; 5 × 105 of K562) were incubated with various MytiLec concentrations as shown. Error bars: SE (n = 3); (B) Annexin V-binding and propidium iodide (PI) incorporation in MytiLec-treated cells. Horizontal axis: binding of FITC-labeled annexin V. Phosphatidylserine externalization and PI incorporation were evaluated by FACS analysis using MEBCYTO apoptosis kit. Ramos (a,c) and K562 (b) cells were treated with MytiLec (a,b: 20 μg/mL; c: 0 μg/mL) for 30 min at 4 °C. Data shown are mean values with error bars = SD of triplicate experiments. Asterisks = significant differences (p < 0.05) between treated and control groups. Full article ">Figure 3
Internalization of FITC-conjugated MytiLec into Burkitt’s lymphoma cells. Incubation time: 0 min (a,b); and 2 h (c–f). Cells are treated with FITC-MytiLec in the presence of 25 mM D-galactose, as negative control (e,f). Cells were observed by fluorescence (a,c,e; ex 498 nm and em 522 nm) and phase-contrast (b,d,f), respectively. Arrows in c and d indicated shrunken cells. Bars indicated 50 μm. Full article ">Figure 4
Effects of MytiLec treatment on MEK, ERK, and cell cycle-related molecules in Burkitt’s lymphoma Ramos cells. (A) Phosphorylation and expression levels of MEK1/2, ERK1/2 and p21, p27, CDK6 and cyclinD3 were shown, respectively. Cells (4 × 105 in each experiment) were treated with various concentrations of MytiLec as shown, and activation levels were evaluated by Western blotting of lysates. Solid and dotted lines indicated increasing and decreasing trends, respectively. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; (B) Relative densitometric quantification of P-MEK/MEK, P-ERK/ERK and p21/GAPDH. Each experiment was repeated three times. Full article ">Figure 5
Phosphorylation of JNK and p38 kinase by MytiLec in Burkitt's lymphoma Ramos. Cells (5 × 105) were treated with (+) or without (−) 20 μg/mL MytiLec, and phosphorylation was evaluated by Western-blotting of cell lysates. (A) P-ERK, P-JNK, and P-p38: phosphorylated forms of ERK, JNK, and p38 kinase, respectively. Asterisks: increased phosphorylation; (B) Relative densitometric quantification of P-ERK/ERK, P-JNK/JNK and P-p38/p38. Each experiment was repeated three times. Full article ">Figure 6
Treatment with MEK inhibitor U0126 reversed up-regulation of p21 expression mediated by MytiLec-induced phosphorylation of MEK-ERK pathway. (A) Burkitt's lymphoma Ramos cells (4 × 105) were treated with (+) or without (−) 20 μM U0126 for 5 h, and then with 20 μg/mL MytiLec. Expression levels of P-MEK1/2, P-ERK1/2, and p21 were evaluated by Western-blotting. Asterisks: disappearance of signals; (B) Relative densitometric quantification of P-MEK, P-ERK and p21 with (U0126/MytiLec) or without (MytiLec) the MEK inhibitor. Each experiment was repeated three times. Full article ">Figure 7
Activation by MytiLec of procaspase-3, procaspase-9, and TNF-α. (A) Activation of procaspase-3 in Ramos (4 × 105 cells) incubated with various concentrations of MytiLec as shown, evaluated by Western blotting; (B) Up-regulation of TNF-α by MytiLec, and inhibition of caspase activation and TNF-α expression by P-MEK inhibitor U0126 and caspase-3 inhibitor Zn-DEVD-FMK (DEVD). Ramos (4 × 105 cells) were treated with (+) or without (−) 10 μM U0126 or 10 μM DEVD for 2 h, and then with 20 μg/mL MytiLec. TNF-α expression and caspase-3/-9 activation were evaluated by Western blotting. Solid and dotted lines indicated increasing and decreasing trends of phosphorylation, respectively. Asterisks: disappearance of signals. Each experiment was repeated three times. Full article ">

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Open AccessReview

Potential Threats Posed by Tetrodotoxins in UK Waters: Examination of Detection Methodology Used in Their Control

by Andrew D. Turner, Cowan Higgins, Wendy Higman and James Hungerford

Mar. Drugs 2015, 13(12), 7357-7376; https://doi.org/10.3390/md13127070 - 11 Dec 2015

Cited by 29 | Viewed by 7742

Abstract

Tetrodotoxin is a neurotoxin responsible for many human fatalities, most commonly following the consumption of pufferfish. Whilst the source of the toxin has not been conclusively proven, it is thought to be associated with various species of marine bacteria. Whilst the toxins are [...] Read more.

Tetrodotoxin is a neurotoxin responsible for many human fatalities, most commonly following the consumption of pufferfish. Whilst the source of the toxin has not been conclusively proven, it is thought to be associated with various species of marine bacteria. Whilst the toxins are well studied in fish and gastropods, in recent years, there have been a number of reports of tetrodotoxin occurring in bivalve shellfish, including those harvested from the UK and other parts of Europe. This paper reviews evidence concerning the prevalence of tetrodotoxins in the UK together with methodologies currently available for testing. Biological, biomolecular and chemical methods are reviewed, including recommendations for further work. With the recent development of quantitative chromatographic methods for these and other hydrophilic toxins, as well as the commercial availability of rapid testing kits, there are a number of options available to ensure consumers are protected against this threat. Full article

(This article belongs to the Special Issue Emerging Marine Toxins)

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1075 KiB

Open AccessArticle

Carotenoids, Phenolic Compounds and Tocopherols Contribute to the Antioxidative Properties of Some Microalgae Species Grown on Industrial Wastewater

by Hamed Safafar, Jonathan Van Wagenen, Per Møller and Charlotte Jacobsen

Mar. Drugs 2015, 13(12), 7339-7356; https://doi.org/10.3390/md13127069 - 11 Dec 2015

Cited by 335 | Viewed by 14951

Abstract

This study aimed at investigating the potential of microalgae species grown on industrial waste water as a new source of natural antioxidants. Six microalgae from different classes, including Phaeodactylum sp. (Bacillariophyceae), Nannochloropsis sp. (Eustigmatophyceae), Chlorella sp., Dunaniella sp., and Desmodesmus sp. (Chlorophyta), were [...] Read more.

This study aimed at investigating the potential of microalgae species grown on industrial waste water as a new source of natural antioxidants. Six microalgae from different classes, including Phaeodactylum sp. (Bacillariophyceae), Nannochloropsis sp. (Eustigmatophyceae), Chlorella sp., Dunaniella sp., and Desmodesmus sp. (Chlorophyta), were screened for their antioxidant properties using different in vitro assays. Natural antioxidants, including pigments, phenolics, and tocopherols, were measured in methanolic extracts of microalgae biomass. Highest and lowest concentrations of pigments, phenolic compounds, and tocopherols were found in Desmodesmus sp. and Phaeodactylum tricornuotom microalgae species, respectively. The results of each assay were correlated to the content of natural antioxidants in microalgae biomass. Phenolic compounds were found as major contributors to the antioxidant activity in all antioxidant tests while carotenoids were found to contribute to the 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging activity, ferrous reduction power (FRAP), and ABTS-radical scavenging capacity activity. Desmodesmus sp. biomass represented a potentially rich source of natural antioxidants, such as carotenoids (lutein), tocopherols, and phenolic compounds when cultivated on industrial waste water as the main nutrient source. Full article

(This article belongs to the Special Issue Green Chemistry Approach to Marine Products)

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1161 KiB

Open AccessFeature PaperReview

Genipin-Crosslinked Chitosan Gels and Scaffolds for Tissue Engineering and Regeneration of Cartilage and Bone

by Riccardo A. A. Muzzarelli, Mohamad El Mehtedi, Carlo Bottegoni, Alberto Aquili and Antonio Gigante

Mar. Drugs 2015, 13(12), 7314-7338; https://doi.org/10.3390/md13127068 - 11 Dec 2015

Cited by 219 | Viewed by 20639

Abstract

The present review article intends to direct attention to the technological advances made since 2009 in the area of genipin-crosslinked chitosan (GEN-chitosan) hydrogels. After a concise introduction on the well recognized characteristics of medical grade chitosan and food grade genipin, the properties of [...] Read more.

The present review article intends to direct attention to the technological advances made since 2009 in the area of genipin-crosslinked chitosan (GEN-chitosan) hydrogels. After a concise introduction on the well recognized characteristics of medical grade chitosan and food grade genipin, the properties of GEN-chitosan obtained with a safe, spontaneous and irreversible chemical reaction, and the quality assessment of the gels are reviewed. The antibacterial activity of GEN-chitosan has been well assessed in the treatment of gastric infections supported by Helicobacter pylori. Therapies based on chitosan alginate crosslinked with genipin include stem cell transplantation, and development of contraction free biomaterials suitable for cartilage engineering. Collagen, gelatin and other proteins have been associated to said hydrogels in view of the regeneration of the cartilage. Viability and proliferation of fibroblasts were impressively enhanced upon addition of poly-l-lysine. The modulation of the osteocytes has been achieved in various ways by applying advanced technologies such as 3D-plotting and electrospinning of biomimetic scaffolds, with optional addition of nano hydroxyapatite to the formulations. A wealth of biotechnological advances and know-how has permitted reaching outstanding results in crucial areas such as cranio-facial surgery, orthopedics and dentistry. It is mandatory to use scaffolds fully characterized in terms of porosity, pore size, swelling, wettability, compressive strength, and degree of acetylation, if the osteogenic differentiation of human mesenchymal stem cells is sought: in fact, the novel characteristics imparted by GEN-chitosan must be simultaneously of physico-chemical and cytological nature. Owing to their high standard, the scientific publications dated 2010–2015 have met the expectations of an interdisciplinary audience. Full article

(This article belongs to the Special Issue Advances in Marine Chitin and Chitosan II, 2017)

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Genipin crosslinks chitosan spontaneously at a quite small molar ratio. On the right, two chitosan chains (represented by their structural units) react covalently with one mole of genipin to yield two newly formed chemical functions, namely the monosubstituted amide and the tertiary amine. Full article ">Figure 2
13C NMR spectrum of chitosan film crosslinked with genipin 0.10%. At 23.0 ppm the resonance signal of alkyl groups in the crosslinked chitosan was attributed to the chitosan + genipin linkage. The signal at 170.5 ppm, assigned to the ester group of plain genipin, disappeared as a consequence of the reaction, thus the resonance at 181.3 ppm is assigned to the amide generated by the reaction between the amino group of chitosan and the ester group of genipin. Full article ">Figure 3
Anti-inflammatory effect of genipin + glycine blue pigment on edema in mice. Maximum edema depression was observed 1 h after edema induction. Notably, treatment with blue pigments at 120 mg/kg reduced edema by ca. 22% (from ca. 60% to 38%) at 1 h, whereas the positive control, dexamethasone (10 mg/kg) depressed the edema by ca. 35% at 1 h. The data are indicative of the safety of genipin which alleviates inflammation by exerting biochemical actions favorable to the organism. Reproduced from [<a href="#B33-marinedrugs-13-07068" class="html-bibr">33</a>]. Full article ">Figure 4
Chitosan-protein scaffolds crosslinked with genipin at various concentrations, under identical conditions. The intensity of the blue color is an indication of the extent of crosslinking. Full article ">Figure 5
Typical genipin-crosslinked chitosan fiber mat obtained from electrospun chitosan mat subsequently treated with genipin: the nanofibrous form is preserved. Full article ">

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Open AccessArticle

Long-Term Feeding of Chitosan Ameliorates Glucose and Lipid Metabolism in a High-Fructose-Diet-Impaired Rat Model of Glucose Tolerance

by Shing-Hwa Liu, Fang-Ying Cai and Meng-Tsan Chiang

Mar. Drugs 2015, 13(12), 7302-7313; https://doi.org/10.3390/md13127067 - 10 Dec 2015

Cited by 29 | Viewed by 6314

Abstract

This study was designed to investigate the effects of long-term feeding of chitosan on plasma glucose and lipids in rats fed a high-fructose (HF) diet (63.1%). Male Sprague-Dawley rats aged seven weeks were used as experimental animals. Rats were divided into three groups: [...] Read more.

This study was designed to investigate the effects of long-term feeding of chitosan on plasma glucose and lipids in rats fed a high-fructose (HF) diet (63.1%). Male Sprague-Dawley rats aged seven weeks were used as experimental animals. Rats were divided into three groups: (1) normal group (normal); (2) HF group; (3) chitosan + HF group (HF + C). The rats were fed the experimental diets and drinking water ad libitum for 21 weeks. The results showed that chitosan (average molecular weight was about 3.8 × 10⁵ Dalton and degree of deacetylation was about 89.8%) significantly decreased body weight, paraepididymal fat mass, and retroperitoneal fat mass weight, but elevated the lipolysis rate in retroperitoneal fats of HF diet-fed rats. Supplementation of chitosan causes a decrease in plasma insulin, tumor necrosis factor (TNF)-α, Interleukin (IL)-6, and leptin, and an increase in plasma adiponectin. The HF diet increased hepatic lipids. However, intake of chitosan reduced the accumulation of hepatic lipids, including total cholesterol (TC) and triglyceride (TG) contents. In addition, chitosan elevated the excretion of fecal lipids in HF diet-fed rats. Furthermore, chitosan significantly decreased plasma TC, low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), the TC/high-density lipoprotein cholesterol (HDL-C) ratio, and increased the HDL-C/(LDL-C + VLDL-C) ratio, but elevated the plasma TG and free fatty acids concentrations in HF diet-fed rats. Plasma angiopoietin-like 4 (ANGPTL4) protein expression was not affected by the HF diet, but it was significantly increased in chitosan-supplemented, HF-diet-fed rats. The high-fructose diet induced an increase in plasma glucose and impaired glucose tolerance, but chitosan supplementation decreased plasma glucose and improved impairment of glucose tolerance and insulin tolerance. Taken together, these results indicate that supplementation with chitosan can improve the impairment of glucose and lipid metabolism in a HF-diet-fed rat model. Full article

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The changes of body weight in normal and fructose rats fed different experimental diets for 21 weeks in prophylactic experiment. Results are expressed as mean ± SD for n = 8 rats per group. Normal: normal control, HF: high fructose, HF + CS: high-fructose + chitosan (prophylactic experiment) * p < 0.05 compared with H by independent-samples t-test. Full article ">Figure 2
The changes of plasma glucose concentration after oral glucose tolerance test (OGTT) (A) and insulin tolerance test (ITT) (B) performed in rats fed the different experimental diets for 16 and 18 weeks, respectively. Results are expressed as mean ± SD for n = 8 rats per each group. Normal: normal control; HF: high fructose; HF + CS: high fructose + chitosan; * p < 0.05 compared with H by independent-samples t-test. Full article ">Figure 3
The change of soleus muscle glucose transporter 4 (GLUT4) in normal and high-fructose rats fed the different experimental diets. Results are expressed as mean ± SD for n = 8 rats per each group. Normal: normal control, HF : high fructose, HF + CS: high fructose + chitosan; * p < 0.05 compared with H by independent-samples t-test. Full article ">Figure 4
The effect of hepatic Fatty acid synthase (FAS) and Acyl-CoA crboxylase (ACC) in normal and high-fructose rats fed the different experimental diets. Results are expressed as mean ± SD for n = 8 rats per each group. Normal: normal control, HF : high fructose, HF + CS: high fructose + chitosan * p < 0.05 compared with H by independent-samples t-test. Full article ">Figure 5
The change of plasma Angptl4 /α-tubulin ratio in normal and high-fructose rats fed the different experimental diets. Results are expressed as mean ± SD for n = 8 rats per each group. Normal: normal control, HF : high fructose, HF + CS: high fructose + chitosan. * p < 0.05 compared with HF by independent-sample’s t-test. Full article ">Figure 6
The changes of lipolysis rate of retroperitoneal and epididymal fat pads in normal and high fructose rats fed the different experimental diets. Results are expressed as mean ± SD for n = 8 rats per each group. Normal: normal control, HF: high fructose, HF+CS: high fructose + chitosan * p < 0.05 compared with HF by independent-samples t-test. Full article ">

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Open AccessCorrection

Correction: Vinayak, V., et al. Diatom Milking: A Review and New Approaches. Marine Drugs 2015, 13, 2629–2665

by Vandana Vinayak, Kalina M. Manoylov, Hélène Gateau, Vincent Blanckaert, Josiane Hérault, Gaëlle Pencréac’h, Justine Marchand, Richard Gordon and Benoît Schoefs

Mar. Drugs 2015, 13(12), 7301; https://doi.org/10.3390/md13127063 - 8 Dec 2015

Cited by 1 | Viewed by 3534

Abstract

n/a Full article

644 KiB

Open AccessArticle

Isolation and Synthesis of Laxaphycin B-Type Peptides: A Case Study and Clues to Their Biosynthesis

by Louis Bornancin, France Boyaud, Zahia Mahiout, Isabelle Bonnard, Suzanne C. Mills, Bernard Banaigs and Nicolas Inguimbert

Mar. Drugs 2015, 13(12), 7285-7300; https://doi.org/10.3390/md13127065 - 5 Dec 2015

Cited by 22 | Viewed by 6869

Abstract

The laxaphyci’s B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the [...] Read more.

The laxaphyci’s B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the beta position, and all containing a rare beta-amino decanoic acid. Nevertheless, they can be differentiated due to slight variations in the composition of their amino acids, but the configuration of their alpha carbon remains conserved. Here, we provide the synthesis and characterization of new laxaphycin B-type peptides. In doing so we discuss how the synthesis of laxaphycin B and analogues was developed. We also isolate minor acyclic laxaphycins B, which are considered clues to their biosynthesis. Full article

(This article belongs to the Special Issue Compounds from Cyanobacteria)

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471 KiB

Open AccessFeature PaperArticle

Biotechnological Production of Docosahexaenoic Acid Using Aurantiochytrium limacinum: Carbon Sources Comparison And Growth Characterization

by Sergi Abad and Xavier Turon

Mar. Drugs 2015, 13(12), 7275-7284; https://doi.org/10.3390/md13127064 - 5 Dec 2015

Cited by 29 | Viewed by 8487

Abstract

Aurantiochytrium limacinum, a marine heterotrophic protist/microalga has shown interesting yields of docosahexaenoic acid (DHA) when cultured with different carbon sources: glucose, pure and crude glycerol. A complete study in a lab-scale fermenter allowed for the characterization and comparison of the growth kinetic [...] Read more.

Aurantiochytrium limacinum, a marine heterotrophic protist/microalga has shown interesting yields of docosahexaenoic acid (DHA) when cultured with different carbon sources: glucose, pure and crude glycerol. A complete study in a lab-scale fermenter allowed for the characterization and comparison of the growth kinetic parameters corresponding to each carbon source. Artificial Marine Medium (AMM) with glucose, pure and crude glycerol offered similar biomass yields. The net growth rates (0.10–0.12 h⁻¹), biomass (0.7–0.8 g cells/g Substrate) and product (0.14–0.15 g DHA/g cells) yields, as well as DHA productivity were similar using the three carbon sources. Viable potential applications to valorize crude glycerol are envisioned to avoid an environmental problem due to the excess of byproduct. Full article

(This article belongs to the Special Issue Marine Lipids)

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1254 KiB

Open AccessArticle

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3 ^‡

by Megumi Yamagishi, Ritsuko Hosoda-Yabe, Hideki Tamai, Miku Konishi, Akihiro Imamura, Hideharu Ishida, Tomio Yabe, Hiromune Ando and Makoto Kiso

Mar. Drugs 2015, 13(12), 7250-7274; https://doi.org/10.3390/md13127062 - 5 Dec 2015

Cited by 18 | Viewed by 6948

Abstract

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically [...] Read more.

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling. Full article

(This article belongs to the Special Issue Marine Glycoconjugates)

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1261 KiB

Open AccessConference Report

The 9th European Conference on Marine Natural Products

by RuAngelie Edrada-Ebel and Marcel Jaspars

Mar. Drugs 2015, 13(12), 7150-7249; https://doi.org/10.3390/md13127059 - 3 Dec 2015

Cited by 5 | Viewed by 11160

Abstract

The 9th European Conference on Marine Natural Products (ECMNP) in Glasgow follows its predecessors in La Toja (2013), Tjärnö (2011), Porto (2009), Ischia (2007), Paris (2005), Elmau (2002), Santiago de Compostela (1999), and Athens (1997). [...] Full article

(This article belongs to the Special Issue Selected Papers from the 9th European Conference on Marine Natural Products)

1103 KiB

Open AccessArticle

Briarenolides U–Y, New Anti-Inflammatory Briarane Diterpenoids from an Octocoral Briareum sp. (Briareidae)

by Yin-Di Su, Tung-Ying Wu, Zhi-Hong Wen, Ching-Chyuan Su, Yu-Hsin Chen, Yu-Chia Chang, Yang-Chang Wu, Jyh-Horng Sheu and Ping-Jyun Sung

Mar. Drugs 2015, 13(12), 7138-7149; https://doi.org/10.3390/md13127060 - 3 Dec 2015

Cited by 10 | Viewed by 4872

Abstract

Five new 13,14-epoxybriarane diterpenoids, briarenolides U–Y (1–5), were isolated from the octocoral Briareum sp. The structures of briaranes 1–5 were elucidated by spectroscopic methods. Briarenolides U–Y (1–5) were found to significantly inhibit the [...] Read more.

Five new 13,14-epoxybriarane diterpenoids, briarenolides U–Y (1–5), were isolated from the octocoral Briareum sp. The structures of briaranes 1–5 were elucidated by spectroscopic methods. Briarenolides U–Y (1–5) were found to significantly inhibit the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Full article

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The structures of briarenolides U–Y (1–5) and briaexcavatolide N (6). Full article ">Figure 2
The computer-generated model of 1 using MM2 force field calculations and the calculated distances (Å) between selected protons with key NOESY correlations. Full article ">Figure 3
The computer-generated model of 5 using MM2 force field calculations and the calculated distances (Å) between selected protons with key NOESY correlations. Full article ">Figure 4
Effects of compounds briarenolides U–Y (1–5) on pro-inflammatory iNOS and COX-2 protein expression in the LPS-stimulated murine macrophage cell line RAW264.7. (A) The relative density of iNOS immunoblot; (B) the relative density of COX-2 immunoblot. The relative intensity of the LPS-stimulated group was taken to be 100%. Band intensities were quantified by densitometry and are indicated as the percent change relative to that of the LPS-stimulated group. Briarenolides U–Z (1–5) and dexamethasone (Dex) significantly inhibited LPS-induced iNOS and COX-2 protein expression in macrophages. The experiments were repeated three times (* p < 0.05, significantly different from the LPS-stimulated group). Full article ">

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Open AccessArticle

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata

by Leila Basti, Hajime Uchida, Ryoji Matsushima, Ryuichi Watanabe, Toshiyuki Suzuki, Toshifumi Yamatogi and Satoshi Nagai

Mar. Drugs 2015, 13(12), 7124-7137; https://doi.org/10.3390/md13127061 - 3 Dec 2015

Cited by 26 | Viewed by 6637

Abstract

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature [...] Read more.

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell⁻¹·day⁻¹) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (R_tox) was not affected by temperature. During EG, the average specific toxin production rate (µ_tox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day⁻¹ at 30 °C, and then decreased. Over the entire growth span, µ_tox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µ_tox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth. Full article

(This article belongs to the Special Issue Okadaic Acid and Dinophysis Toxins)

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Figure 1
Changes in cell density of Dinophysis caudata (a), fed with Mesodinium rubrum (b), grown under different temperatures. Vertical bars denote the standard deviation (SD) of the mean (n = 3). Full article ">Figure 2
Average specific growth rates of Dinophysis caudata during the exponential growth phase under different experimental temperatures. Vertical bars denote the standard deviation (SD) of the mean (n = 3). Different letters indicate significant differences among treatments (ANOVA, Neuwman-Keuls, p < 0.05). Full article ">Figure 3
Concentration of PTX-2 (ng·mL−1) and cellular production (particulate plus dissolved) of PTX-2 (pg·cell−1) in cultures of Dinophysis caudata grown at different temperatures. Vertical bars denote the standard deviation (SD) of the mean (n = 3). PTX = pectenotoxins. Full article ">Figure 4
Net toxin production (Rtox) of PTX2 by Dinophysis caudata grown at different temperatures. Vertical bars denote the standard deviation (SD) of the mean (n = 3). Full article ">Figure 5
Average specific toxin production rate (µtox) of PTX-2 by Dinophysis caudata during the exponential growth phase, cultured under different temperatures. Vertical bars denote the standard deviation (SD) of the mean (n = 3). Full article ">Figure 6
Relationship between specific toxin production rate (µtox) and specific growth rate (µ) of Dinophysis caudata during the exponential growth phase. Averaged values were plotted. Full article ">

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Open AccessArticle

Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin

by Heidi Wichmann, Farina Vocke, Thorsten Brinkhoff, Meinhard Simon and Christiane Richter-Landsberg

Mar. Drugs 2015, 13(12), 7113-7123; https://doi.org/10.3390/md13127058 - 27 Nov 2015

Cited by 11 | Viewed by 6170

Abstract

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in [...] Read more.

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3–0.5 µg/mL (1.4–2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca²⁺-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death. Full article

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Cytotoxic effects of tropodithietic acid (TDA) in OLN-93 and N2a cells after incubation with tropodithietic acid (TDA) for 24 h. (A) Effect of TDA on cell morphology. Hoffman modulation contrast images are shown. OLN-93 and N2a cells were either treated with dimethyl sulfoxide as negative control (Co) or subjected to 0.3 µg/mL (1.4 µM) or 0.5 µg/mL (2.4 µM) TDA. Scale bar 50 µm. (B) MTT (thiazolyl blue tetrazolium bromide) assay. Cells were incubated with increasing TDA concentrations (0.1–1 µg/mL, as indicated) (OLN-93 dark grey bars, N2a cells light grey bars). (C) Western blot analysis. Cell lysates of OLN-93 and N2a cells were prepared and subjected to immunoblot analysis using antibodies as indicated on the right (for details see <a href="#sec3-marinedrugs-13-07058" class="html-sec">Experimental Section</a>). Quantitative evaluation of the immunoblots was carried out by densitometric scanning and Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA). Activated ERK 1/2 (ERK 1/2 P) is expressed as percentage of the total amount of ERK1/2 (100%). Hsp32 is expressed as percentage of glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 100%), which was used as loading control (100%). <a href="#marinedrugs-13-07058-f001" class="html-fig">Figure 1</a>B,C: Statistical evaluation was carried out by students t-test: * p < 0.05 significant and ** p < 0.001 highly significant compared to the control. Full article ">Figure 2
Effect of TDA on microtubule organization and mitochondrial integrity. Cells were subjected to 0.3 µg/mL TDA for 24 h. Subsequently, cells were incubated for 30 min with MitoTrackerRed, fixed with methanol and subjected to indirect immunofluorescence using antibodies against α-tubulin (green). Nuclei were stained with DAPI (blue). Co, untreated control incubated with the solvent DMSO only. Lower panel, OLN-93 cells shown at a higher magnification. Scale bars: 20 µm. Full article ">Figure 3
Effect of TDA on mitochondrial integrity. Cells were subjected to TDA for 24 h as indicated. Thereafter, cells were subjected to indirect immunofluorescence using antibodies against heat shock protein 60 (Hsp60), or incubated for 30 min with MitoTrackerRed or MitoTrackerGreen. Co, untreated control incubated with the solvent DMSO only. Scale bar: 20 µm. Full article ">Figure 4
Fura-2 AM calcium imaging. TDA induces an increase of intracellular Ca2+ in N2a cells. (a–c) Cells were incubated with Fura-2-AM solution at 37 °C in the dark. Subsequently, 0.1 µg/mL TDA was added and cells were monitored after 10 min, 35 min and 48 min, as indicated. Scale bar: 50 µm. (d–f) Enlargement of the insets depicted in the upper panels (red arrows: necrotic cells). Scale bars: 20 µm. (g–i) Negative control without TDA additive. Scale bars: 20 µm. Full article ">Figure 5
Effect of TDA on F-actin cytoskeleton in OLN-93 cells. Cells were subjected to 0.1 µg/mL TDA for 24 h. Subsequently, cells were fixed with 3% paraformaldehyde, incubated with phalloidin green and indirect immunofluorescence using antibodies against α-tubulin (red) was carried out. Nuclei were stained with DAPI (blue). Co, untreated control. Scale bars: 50 µm (upper two images, 200× magnification), 20 µm (1000× magnification). Full article ">

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Open AccessReview

Potential Threats Posed by New or Emerging Marine Biotoxins in UK Waters and Examination of Detection Methodologies Used for Their Control: Cyclic Imines

by Keith Davidson, Clothilde Baker, Cowan Higgins, Wendy Higman, Sarah Swan, Andrea Veszelovszki and Andrew D. Turner

Mar. Drugs 2015, 13(12), 7087-7112; https://doi.org/10.3390/md13127057 - 26 Nov 2015

Cited by 38 | Viewed by 6381

Abstract

Cyclic imines (CIs) are a group of phytoplankton produced toxins related to shellfish food products, some of which are already present in UK and European waters. Their risk to shellfish consumers is poorly understood, as while no human intoxication has been definitively related [...] Read more.

Cyclic imines (CIs) are a group of phytoplankton produced toxins related to shellfish food products, some of which are already present in UK and European waters. Their risk to shellfish consumers is poorly understood, as while no human intoxication has been definitively related to this group, their fast acting toxicity following intraperitoneal injection in mice has led to concern over their human health implications. A request was therefore made by UK food safety authorities to examine these toxins more closely to aid possible management strategies. Of the CI producers only the spirolide producer Alexandrium ostenfeldii is known to exist in UK waters at present but trends in climate change may lead to increased risk from other organisms/CI toxins currently present elsewhere in Europe and in similar environments worldwide. This paper reviews evidence concerning the prevalence of CIs and CI-producing phytoplankton, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern. Full article

(This article belongs to the Special Issue Emerging Marine Toxins)

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Figure 1

Figure 1
Structure of some of the known spirolides [<a href="#B17-marinedrugs-13-07057" class="html-bibr">17</a>,<a href="#B20-marinedrugs-13-07057" class="html-bibr">20</a>]. Full article ">Figure 2
Chemical structure of Gymnodimines (A) GYM-A; (B) GYM-B; (C) GYM-C [<a href="#B31-marinedrugs-13-07057" class="html-bibr">31</a>]; and (D) 12-methylgymnodimine [<a href="#B32-marinedrugs-13-07057" class="html-bibr">32</a>]. Full article ">Figure 3
Chemical structure of known pinnatoxins, modified from [<a href="#B40-marinedrugs-13-07057" class="html-bibr">40</a>,<a href="#B45-marinedrugs-13-07057" class="html-bibr">45</a>]. Full article ">Figure 4
Chemical structure of known pteriatoxins [<a href="#B47-marinedrugs-13-07057" class="html-bibr">47</a>]. Full article ">Figure 5
Planar structure of the known prorocentrolides. Full article ">Figure 6
Distribution of cells belonging to the Alexandrium ostenfeldii group in Scottish coastal waters. Identification was confirmed at most locations by examination of the thecal plates, but it is likely that the species is much more widespread. Full article ">

1180 KiB

Open AccessArticle

Effects of Organic and Inorganic Nitrogen on the Growth and Production of Domoic Acid by Pseudo-nitzschia multiseries and P. australis (Bacillariophyceae) in Culture

by Véronique Martin-Jézéquel, Guillaume Calu, Leo Candela, Zouher Amzil, Thierry Jauffrais, Véronique Séchet and Pierre Weigel

Mar. Drugs 2015, 13(12), 7067-7086; https://doi.org/10.3390/md13127055 - 26 Nov 2015

Cited by 37 | Viewed by 7779

Abstract

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in [...] Read more.

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment. Full article

(This article belongs to the Special Issue Metabolites in Diatoms)

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2177 KiB

Open AccessArticle

Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

by Yun-Hee Choi, Dong Joo Yang, Atul Kulkarni, Sang Hyun Moh and Ki Woo Kim

Mar. Drugs 2015, 13(12), 7055-7066; https://doi.org/10.3390/md13127056 - 26 Nov 2015

Cited by 41 | Viewed by 7604

Abstract

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved [...] Read more.

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. Full article

(This article belongs to the Special Issue Marine Secondary Metabolites)

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Full article ">Figure 1
Characterization of MAAs (mycosporine-like amino acids): (A) HPLC-DAD (330 nm) chromatograms of SH, P334, and M-Gly. The retention time for SH, M-Gly, and P334 was 4.395, 5.356, and 6.252 minutes, respectively; (B) Triplequadruple ESI-MS/MS spectra of SH, P334, and M-Gly. Protonated parent molecule is indicated in bold type for each MAA (inset structure of respective MAA). Full article ">Figure 2
Wound healing effects of MAAs: (A–C) HaCaT cells were treated with indicated concentrations of M-Gly (A); P334 (B); or SH (C) for 24 h and cell viability was assessed by MTT assay; (D) HaCaT cells were scratched using pipet tips and incubated for 24 h in the presence of vehicle (t = 24, b), EGF (100 ng/mL, c), M-Gly (0.1 mg/mL, d), P334 (0.05 mg/mL, e), and SH (0.05 mg/mL, f). The t = 0 (a) indicates the time for scratching; (E) Percent (%) of healing area. Values are mean ± standard error of the mean (SEM). (* p < 0.05, one-way ANOVA followed by Bonferroni’s post hoc test for A to C and Student’s t-test for E). Full article ">Figure 3
Activation of FAK (focal adhesion kinases), ERK(extracellular signal-regulated kinases), and JNK (c-Jun N-terminal kinases) by MAAs: (A) Activation of FAK, ERK, and JNK by treatment of EGF (100 ng/mL), M-Gly (0.1 mg/mL), and P334 (0.05 mg/mL), and SH (0.05 mg/mL) in HaCaT cells; (B) The intensity of each band was measured with densitometer and expressed as protein level normalized to GAPDH; (C) Effect of ERK (PD98059; 50 μM) and FAK (FAK14; 5 μM) inhibitors; (D) Effect of SP600125 (10 μM), a JNK inhibitor. GAPDH was used as an internal control. Values represent mean ± SEM (* p < 0.05, Student’s t-test). Full article ">Figure 4
Inhibition of ERK and JNK impaired the MAAs-mediated wound healing effects. (A–B) Wound healing effects mediated by indicated MAAs was blunted by pretreatment of ERK inhibitor (PD98059; 50 μM). Scratched cells (a; t = 0) were incubated with DMSO (b), M-Gly (c), P334 (d), or SH (e) for 24 h. ERK inhibitor was pretreated for 2 h prior to incubation with M-Gly (f), P334 (g), or SH (h); (C–D) Wound healing effects mediated by indicated MAAs were decreased by pretreatment of JNK inhibitor (SP600125; 10 μM). Wounded cells (a; t = 0) were treated with DMSO (b), M-Gly (c), P334 (d), or SH (e) for 24 h. JNK inhibitor was pretreated for 2 h prior to incubation with M-Gly (f), P334 (g), or SH (h). The area of the wound was measured and the percentage of healed area calculated. Values represent mean ± SEM (* p < 0.05, Student’s t-test). Full article ">

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Mar. Drugs, Volume 13, Issue 12 (December 2015) – 22 articles

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