[go: up one dir, main page]
More Web Proxy on the site http://driver.im/
Next Issue
Volume 16, February
Previous Issue
Volume 15, December
You seem to have javascript disabled. Please note that many of the page functionalities won't work as expected without javascript enabled.
 
 
ijms-logo

Journal Browser

Journal Browser

Int. J. Mol. Sci., Volume 16, Issue 1 (January 2015) – 130 articles , Pages 1-2268

  • Issues are regarded as officially published after their release is announced to the table of contents alert mailing list.
  • You may sign up for e-mail alerts to receive table of contents of newly released issues.
  • PDF is the official format for papers published in both, html and pdf forms. To view the papers in pdf format, click on the "PDF Full-text" link, and use the free Adobe Reader to open them.
Cover Story:
Order results
Result details
Section
Select all
Export citation of selected articles as:
5297 KiB  
Article
Inhibition of the IgE-Mediated Activation of RBL-2H3 Cells by TIPP, a Novel Thymic Immunosuppressive Pentapeptide
by Qianqian Lian, Yanna Cheng, Chuanqing Zhong and Fengshan Wang
Int. J. Mol. Sci. 2015, 16(1), 2252-2268; https://doi.org/10.3390/ijms16012252 - 20 Jan 2015
Cited by 24 | Viewed by 8566
Abstract
TIPP is a novel thymic immunosuppressive pentapeptide originally obtained from calf thymic immunosuppressive extract. The present study aimed to investigate the inhibitory activity of TIPP on IgE-mediated activation of RBL-2H3 cells. Release of ?-hexosaminidase and histamine, intracellular calcium, membrane ruffling, mRNA levels of [...] Read more.
TIPP is a novel thymic immunosuppressive pentapeptide originally obtained from calf thymic immunosuppressive extract. The present study aimed to investigate the inhibitory activity of TIPP on IgE-mediated activation of RBL-2H3 cells. Release of ?-hexosaminidase and histamine, intracellular calcium, membrane ruffling, mRNA levels of cytokines, cyclooxygenase-2 (COX-2) expression, and activation of mitogen-activated protein kinases (MAP kinases) and NF-?B were determined by colorimetric assay, fluorescence spectrophotometer, confocal fluorescence microscope, quantification PCR, and Western blot, respectively. The results showed that TIPP significantly inhibited the degranulation in IgE-antigen complex-stimulated RBL-2H3 cells without cytotoxicity. TIPP significantly suppressed the increase of intracellular calcium and the rearrangement of F-actin, attenuated the transcription of pro-inflammatory cytokines (IL-3, -4, -6, -13, TNF-?, and monocyte chemotactic protein-1 (MCP-1)), and decreased the expression of COX-2. Western blot analysis showed that TIPP had an inhibitory activity on the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and ERK kinase 1/2 (MEK1/2), and inhibited the activation of NF-?B. The data suggested that TIPP effectively suppressed IgE-mediated activation of RBL-2H3 cells via blocking MEK/ERK and NF-?B signaling pathways. Full article
(This article belongs to the Section Biochemistry)
Show Figures

Figure 1

Figure 1
<p>Chemical structure of TIPP.</p>
Full article ">Figure 2
<p>Effect of thymic immunosuppressive pentapeptide (TIPP) on IgE-mediated degranulation in RBL-2H3 cells. (<b>A</b>) Cytotoxicity of TIPP. Results are expressed as mean ± SD (<span class="html-italic">n =</span> 5); (<b>B</b>–<b>D</b>) represent the effects of anti-DNP-IgE (monoclonal anti-dinitrophenyl antibody produced in mouse, IgE isotype) concentration, dinitrophenyl-human serum albumin (DNP-HAS) concentration, and stimulated time on IgE-mediated degranulation in RBL-2H3 cells. The amount of β-hexosaminidase in culture supernatant was determined as a biomarker of degranulation. Supernatant samples treated with 0.1% Triton X-100 (<span class="html-italic">v</span>/<span class="html-italic">v</span>) were used as a maximum of degranulation. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 3); (<b>E</b>,<b>F</b>) represent the effects of TIPP on β-hexosaminidase and histamine release under optimum conditions. Supernatant samples stimulated with IgE-antigen complex and not treated with TIPP were used as a control of 100%. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 3 for β-hexosaminidase determination and <span class="html-italic">n =</span> 6 for histamine determination). Compared to normal, <sup>###</sup><span class="html-italic"> p</span> &lt; 0.001; compared to control (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), <b>*</b><span class="html-italic"> p</span> &lt; 0.05, <b>**</b><span class="html-italic"> p</span> &lt; 0.01, and <b>***</b><span class="html-italic"> p</span> &lt; 0.001. Keto.: ketotifen.</p>
Full article ">Figure 2 Cont.
<p>Effect of thymic immunosuppressive pentapeptide (TIPP) on IgE-mediated degranulation in RBL-2H3 cells. (<b>A</b>) Cytotoxicity of TIPP. Results are expressed as mean ± SD (<span class="html-italic">n =</span> 5); (<b>B</b>–<b>D</b>) represent the effects of anti-DNP-IgE (monoclonal anti-dinitrophenyl antibody produced in mouse, IgE isotype) concentration, dinitrophenyl-human serum albumin (DNP-HAS) concentration, and stimulated time on IgE-mediated degranulation in RBL-2H3 cells. The amount of β-hexosaminidase in culture supernatant was determined as a biomarker of degranulation. Supernatant samples treated with 0.1% Triton X-100 (<span class="html-italic">v</span>/<span class="html-italic">v</span>) were used as a maximum of degranulation. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 3); (<b>E</b>,<b>F</b>) represent the effects of TIPP on β-hexosaminidase and histamine release under optimum conditions. Supernatant samples stimulated with IgE-antigen complex and not treated with TIPP were used as a control of 100%. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 3 for β-hexosaminidase determination and <span class="html-italic">n =</span> 6 for histamine determination). Compared to normal, <sup>###</sup><span class="html-italic"> p</span> &lt; 0.001; compared to control (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), <b>*</b><span class="html-italic"> p</span> &lt; 0.05, <b>**</b><span class="html-italic"> p</span> &lt; 0.01, and <b>***</b><span class="html-italic"> p</span> &lt; 0.001. Keto.: ketotifen.</p>
Full article ">Figure 3
<p>Effect of TIPP on intracellular calcium level. This result is representative of three independent experiments with similar results.</p>
Full article ">Figure 4
<p>Effects of TIPP on the mRNA levels of (<b>A</b>) IL-3; (<b>B</b>) IL-4; (<b>C</b>) IL-6; (<b>D</b>) IL-13; (<b>E</b>) TNF-α; and (<b>F</b>) monocyte chemotactic protein-1 (MCP-1) in IgE-antigen complex-stimulated RBL-2H3 cells. mRNA level of β-actin was used as an internal control. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 4). Compared with normal group, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with control group (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), <b>*</b> <span class="html-italic">p</span> &lt; 0.05, <b>**</b> <span class="html-italic">p</span> &lt; 0.01, and <b>***</b> <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 4 Cont.
<p>Effects of TIPP on the mRNA levels of (<b>A</b>) IL-3; (<b>B</b>) IL-4; (<b>C</b>) IL-6; (<b>D</b>) IL-13; (<b>E</b>) TNF-α; and (<b>F</b>) monocyte chemotactic protein-1 (MCP-1) in IgE-antigen complex-stimulated RBL-2H3 cells. mRNA level of β-actin was used as an internal control. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 4). Compared with normal group, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with control group (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), <b>*</b> <span class="html-italic">p</span> &lt; 0.05, <b>**</b> <span class="html-italic">p</span> &lt; 0.01, and <b>***</b> <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 5
<p>Confocal fluorescence microscope observation of F-actin (labeled with rhodamin-phalloidin) in RBL-2H3 cells. The arrow represents membrane ruffling caused by F-actin rearrangement. (<b>a</b>) IgE-sensitized RBL-2H3 cells stimulated with PBS; (<b>b</b>) IgE-sensitized RBL-2H3 cells stimulated with DNP-HSA for 30 min; (<b>c</b>–<b>f</b>), IgE-sensitized RBL-2H3 cells, pretreated with 50, 200, and 800 μg/mL of TIPP or 20 μg/mL of ketotifen, then stimulated with DNP-HSA for 30 min. Magnification: 63×.</p>
Full article ">Figure 6
<p>(<b>A</b>) Confocal microscopic observation of the binding of TIPP with RBL-2H3 cells. Pictures represent location of TIPP, nucleus, membrane and merged, respectively. Magnification: 63×; (<b>B</b>–<b>D</b>) represent the concentration-, time- and temperature-dependent characterization of the TIPP binding.</p>
Full article ">Figure 7
<p>Western blot diagram (<b>A</b>) and statistical analysis (<b>B</b>) of COX-2 (cyclooxygenase-2) expression in IgE-antigen complex stimulated RBL-2H3 cells; (<b>C</b>) represents the statistical analysis of COX-2 mRNA levels in IgE-antigen complex stimulated RBL-2H3 cells. The Western blot diagram is a representative of three independent experiment diagrams with similar results. Each lane was loaded with 20 μg of total protein. Results are expressed as mean ± SEM (<span class="html-italic">n</span> = 3). Compared with normal group, <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01; compared with control group, <b>*</b> <span class="html-italic">p</span> &lt; 0.05, <b>***</b> <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 8
<p>Effects of TIPP on the activation of mitogen-activated protein (MAP) kinases (<b>A</b>–<b>D</b>) and extracellular signal-regulated protein kinase (ERK) kinase 1/2 (MEK1/2) (<b>E</b>,<b>F</b>) in IgE-antigen complex stimulated RBL-2H3 cells. The Western blot diagram is a representative of three independent experiment diagrams with similar results. Each lane was loaded with 20 μg of total protein. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 3). Compared with Normal group, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with Control group, <b>*</b> <span class="html-italic">p</span> &lt; 0.05, <b>**</b> <span class="html-italic">p</span> &lt; 0.01.</p>
Full article ">Figure 9
<p>Effect of TIPP on the nuclear translocation of NF-κB. The Western blot diagram is a representative of three independent experiment diagrams with similar results. Each lane was loaded with 20 μg of total protein. Results are expressed as mean ± SEM (<span class="html-italic">n =</span> 3). Compared with Normal group, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with Control group, <b>***</b> <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">
1430 KiB  
Article
Mechanical and Anticorrosive Properties of Graphene/Epoxy Resin Composites Coating Prepared by in-Situ Method
by Zhiyi Zhang, Wenhui Zhang, Diansen Li, Youyi Sun, Zhuo Wang, Chunling Hou, Lu Chen, Yang Cao and Yaqing Liu
Int. J. Mol. Sci. 2015, 16(1), 2239-2251; https://doi.org/10.3390/ijms16012239 - 20 Jan 2015
Cited by 153 | Viewed by 14566
Abstract
The graphene nanosheets-based epoxy resin coating (0, 0.1, 0.4 and 0.7 wt %) was prepared by a situ-synthesis method. The effect of polyvinylpyrrolidone/reduced graphene oxide (PVP-rGO) on mechanical and thermal properties of epoxy resin coating was investigated using nanoindentation technique and thermogravimetric analysis, [...] Read more.
The graphene nanosheets-based epoxy resin coating (0, 0.1, 0.4 and 0.7 wt %) was prepared by a situ-synthesis method. The effect of polyvinylpyrrolidone/reduced graphene oxide (PVP-rGO) on mechanical and thermal properties of epoxy resin coating was investigated using nanoindentation technique and thermogravimetric analysis, respectively. A significant enhancement (ca. 213% and 73 °C) in the Young modulus and thermal stability of epoxy resin coating was obtained at a loading of 0.7 wt %, respectively. Furthermore, the erosion resistance of graphene nanosheets-based epoxy resin coating was investigated by electrochemical measurement. The results showed also that the Rrcco (ca. 0.3 mm/year) of graphene nanosheets-based epoxy resin coating was far lower than neat epoxy resin (1.3 mm/year). Thus, this approach provides a novel route for improving erosion resistance and mechanical-thermal stability of polymers coating, which is expected to be used in mechanical-thermal-corrosion coupling environments. Full article
(This article belongs to the Section Materials Science)
Show Figures

Figure 1

Figure 1
<p>(<b>A</b>) Raman spectra; and (<b>B</b>) AFM of GNS-based epoxy resin coating.</p>
Full article ">Figure 2
<p>SEM of GNS-based epoxy resin coating with various content of (<b>A</b>) 0; (<b>B</b>) 0.1 wt %; (<b>C</b>) 0.4 wt %; and (<b>D</b>) 0.7 wt %. The corresponding digital photographs of GNS-based epoxy resin coating are shown in the inset.</p>
Full article ">Figure 3
<p>Load-displacement curves of GNS-based epoxy resin coating with different contents of (A) 0; (B) 0.1 wt %; (C) 0.4 wt %; and (D) 0.7 wt %.</p>
Full article ">Figure 4
<p>Load-displacement curves of GNS-based epoxy resin coating with different contents of (A) 0; (B) 0.1 wt %; (C) 0.4 wt %; and (D) 0.7 wt %.</p>
Full article ">Figure 5
<p>The TGA curves of GNS-based epoxy resin coating with different contents of (A) 0; (B) 0.1 wt %; (C) 0.4 wt %; and (D) 0.7 wt %.</p>
Full article ">Figure 6
<p>The Tafel plots for GNS-based epoxy resin coating with various content of (A) 0; (B) 0.1 wt %; (C) 0.4 wt %; and (D) 0.7 wt %.</p>
Full article ">Figure 7
<p>SEM images of uncoated Zn plate (<b>A</b>) before; and (<b>B</b>) after corrosion, Zn palate protected GNS-based epoxy resin coating with various content of (<b>C</b>) 0; (<b>D</b>) 0.1 wt %; (<b>E</b>) 0.4 wt %; and (<b>F</b>) 0.7 wt %.</p>
Full article ">Figure 8
<p>Synthetic process of GNS-based epoxy resin coating.</p>
Full article ">
1770 KiB  
Article
De Novo Assembly and Characterization of Narrow-Ridged Finless Porpoise Renal Transcriptome and Identification of Candidate Genes Involved in Osmoregulation
by Rui Ruan, Ai-Huan Guo, Yu-Jiang Hao, Jin-Song Zheng and Ding Wang
Int. J. Mol. Sci. 2015, 16(1), 2220-2238; https://doi.org/10.3390/ijms16012220 - 20 Jan 2015
Cited by 18 | Viewed by 8275
Abstract
During the evolutionary transition from land to water, cetaceans have undergone numerous critical challenges, with osmoregulation being the major one. Two subspecies of the narrow-ridged finless porpoise (Neophocaena asiaeorientalis), the freshwater Yangtze finless porpoise (N. a. asiaeorientalis, NAA) and [...] Read more.
During the evolutionary transition from land to water, cetaceans have undergone numerous critical challenges, with osmoregulation being the major one. Two subspecies of the narrow-ridged finless porpoise (Neophocaena asiaeorientalis), the freshwater Yangtze finless porpoise (N. a. asiaeorientalis, NAA) and the marine East Asian finless porpoise (N. a. sunameri, NAS), provide excellent subjects to understand the genetic basis of osmoregulatory divergence between freshwater and marine mammals. The kidney plays an important and well-established role in osmoregulation in marine mammals and thus, herein, we utilized RNA-seq to characterize the renal transcriptome and preliminarily analyze the divergence between the NAA and the NAS. Approximately 48.98 million clean reads from NAS and 49.40 million clean reads from NAA were obtained by RNA-Seq. And 73,449 (NAS) and 68,073 (NAA) unigenes were assembled. Among these annotations, 22,231 (NAS) and 21,849 (NAA) unigenes were annotated against the NCBI nr protein database. The ion channel complex GO term and four pathways were detected as relevant to osmoregulation by GO and KEGG pathway classification of these annotated unigenes. Although the endangered status of the study species prevented analysis of biological replicates, we identified nine differentially expressed genes (DEGs) that may be vital in the osmoregulation of the narrow-ridged finless porpoise and worthwhile for future studies. Of these DEGs, the differential expression and distribution of the aquaporin-2 (AQP2) in the collecting duct were verified using immunohistochemical experiments. Together, this work is the first report of renal transcriptome sequencing in cetaceans, and it will provide a valuable resource for future molecular genetics studies on cetacean osmoregulation. Full article
(This article belongs to the Section Biochemistry)
Show Figures

Figure 1

Figure 1
<p>Distributions of the species similarity in NAA and NAS. NAA: the Yangtze finless porpoise; NAS: the East Asian finless porpoise.</p>
Full article ">Figure 2
<p>KEGG pathway classification of unigenes. (<b>a</b>) NAA; (<b>b</b>) NAS. The KEGG pathways were categorized into five groups, A: Cellular process, B: Environmental information processing, C: Genetic information processing, D: Metabolism and E: Organismal system. KEGG: Kyoto Encyclopedia of Genes and Genomes; NAS: the East Asian finless porpoise; NAA: the Yangtze finless porpoise.</p>
Full article ">Figure 2 Cont.
<p>KEGG pathway classification of unigenes. (<b>a</b>) NAA; (<b>b</b>) NAS. The KEGG pathways were categorized into five groups, A: Cellular process, B: Environmental information processing, C: Genetic information processing, D: Metabolism and E: Organismal system. KEGG: Kyoto Encyclopedia of Genes and Genomes; NAS: the East Asian finless porpoise; NAA: the Yangtze finless porpoise.</p>
Full article ">Figure 3
<p>GO enrichment analysis of DEGs between NAS and NAA. GO: Gene Ontology; DEGs: differentially expressed genes; NAS: the East Asian finless porpoise; NAA: the Yangtze finless porpoise.</p>
Full article ">Figure 4
<p>Distributions of AQP2 protein in the medulla of the renicule in NAS and NAA. The dark yellow color shows that AQP2 protein localized to the collecting ducts. Magnification: 40×. AQP2: aquaporin-2; NAS: the East Asian finless porpoise; NAA: the Yangtze finless porpoise.</p>
Full article ">
1450 KiB  
Article
Identification and Analysis of Differentially-Expressed microRNAs in Japanese Encephalitis Virus-Infected PK-15 Cells with Deep Sequencing
by Yuhan Cai, Ling Zhu, Yuanchen Zhou, Xiao Liu, Xiaowan Liu, Xinqiong Li, Qiaoli Lang, Xiaogai Qiao and Zhiwen Xu
Int. J. Mol. Sci. 2015, 16(1), 2204-2219; https://doi.org/10.3390/ijms16012204 - 20 Jan 2015
Cited by 21 | Viewed by 7633
Abstract
Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, causes acute viral encephalitis with high morbidity and mortality in humans and animals. MicroRNAs (miRNAs) are small noncoding RNAs that are important modulators of the intricate host-pathogen interaction networks. However, our knowledge of the changes [...] Read more.
Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, causes acute viral encephalitis with high morbidity and mortality in humans and animals. MicroRNAs (miRNAs) are small noncoding RNAs that are important modulators of the intricate host-pathogen interaction networks. However, our knowledge of the changes that occur in miRNAs in host cells after JEV infection is still limited. To understand the molecular pathogenesis of JEV at the level of posttranscriptional regulation, we used Illumina deep sequencing to sequence two small RNA libraries prepared from PK-15 cells before and after JEV infection. We identified 522 and 427 miRNAs in the infected and uninfected cells, respectively. Overall, 132 miRNAs were expressed significantly differently after challenge with JEV: 78 were upregulated and 54 downregulated. The sequencing results for selected miRNAs were confirmed with RT-qPCR. GO analysis of the host target genes revealed that these dysregulated miRNAs are involved in complex cellular pathways, including the metabolic pathway, inflammatory response and immune response. To our knowledge, this is the first report of the comparative expression of miRNAs in PK-15 cells after JEV infection. Our findings will underpin further studies of miRNAs’ roles in JEV replication and identify potential candidates for antiviral therapies against JEV. Full article
(This article belongs to the Section Biochemistry)
Show Figures

Figure 1

Figure 1
<p>Length distributions of small RNAs in Japanese encephalitis virus (JEV)-infected and -uninfected PK-15 cells.</p>
Full article ">Figure 2
<p>Comparison of differentially-expressed miRNAs between the JEV-infected and -uninfected cells. The Venn diagram displays the distribution of 565 unique miRNAs across the infected group and uninfected group. The dashed circles indicate the miRNAs that were significantly differentially expressed in the infected group relative to the uninfected group.</p>
Full article ">Figure 3
<p>Chromosomal locations of miRNAs based on the numbers of total miRNAs (detected in the infected and uninfected cells) and differentially-expressed miRNAs. “ND” means that the genome location of the pre-miRNA has not been determined.</p>
Full article ">Figure 4
<p>Validation of miRNA expression by RT-qPCR.</p>
Full article ">Figure 5
<p>GO annotation of the predicted miRNA target genes. The figure shows the GO annotation of the upregulated genes (<b>A</b>) and downregulated genes (<b>B</b>) in biological processes, cellular components and molecular functions.</p>
Full article ">
1467 KiB  
Review
Experimental Confirmation of a Whole Set of tRNA Molecules in Two Archaeal Species
by Yoh-ichi Watanabe and Yutaka Kawarabayasi
Int. J. Mol. Sci. 2015, 16(1), 2187-2203; https://doi.org/10.3390/ijms16012187 - 20 Jan 2015
Cited by 2 | Viewed by 5692
Abstract
Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder) is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire [...] Read more.
Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder) is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire genomic sequences of two Creanarchaeota, Aeropyrum pernix and Sulfolobus tokodaii, were determined approximately 15 years ago. In these genome datasets, 47 and 46 tRNA genes were detected, respectively. Among them, 14 and 24 genes, respectively, were predicted to be interrupted tRNA genes. To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers. Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule. Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea. Full article
(This article belongs to the Special Issue Functions of Transfer RNAs)
Show Figures

Figure 1

Figure 1
<p>Overall strategy used to confirm predicted tRNA molecules in <span class="html-italic">A. pernix</span> K1.</p>
Full article ">Figure 2
<p>Agarose electrophoresis patterns of RT-PCR-amplified fragments. (<b>A</b>) Amplified without RT (reverse transcriptase); (<b>B</b>) Amplified with RT. Lanes 1 to 13 contain fragment that were amplified with primer sets 15–27 summarized in previous work; these primers were described previously [<a href="#B9-ijms-16-02187" class="html-bibr">9</a>]. M: 100 bp ladder marker.</p>
Full article ">Figure 3
<p>Summary of the tRNA introns identified in <span class="html-italic">A. pernix</span> K1. Each label includes the tRNA species, the respective intron positions from the 5' end of the respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.</p>
Full article ">Figure 4
<p>Proposed structures of the unspliced forms of interrupted tRNA molecules in <span class="html-italic">A. pernix</span> K1. Splicing sites and anticodon regions are indicated by short arrows and boxes, respectively. (<b>A</b>) tRNA<sup>Arg</sup>(UCU); (<b>B</b>) tRNA<sup>Asp</sup>(GUC); (<b>C</b>) tRNA<sup>Met</sup>(CAU-1); (<b>D</b>) tRNA<sup>Met</sup>(CAU-2); (<b>E</b>) tRNA<sup>Thr</sup>(CGU); (<b>F</b>) tRNA<sup>Pro</sup>(GGG); (<b>G</b>) tRNA<sup>Cys</sup>(GCA); (<b>H</b>) tRNA<sup>Tyr</sup>(GUA); (<b>I</b>) tRNA<sup>Thr</sup>(UGU-1); (<b>J</b>) tRNA<sup>Trp</sup>(CCA); (<b>K</b>) tRNA<sup>Lys</sup>(CUU); (<b>L</b>) tRNA<sup>Lys</sup>(UUU); (<b>M</b>) tRNA<sup>Pro</sup>(CGG).</p>
Full article ">Figure 4 Cont.
<p>Proposed structures of the unspliced forms of interrupted tRNA molecules in <span class="html-italic">A. pernix</span> K1. Splicing sites and anticodon regions are indicated by short arrows and boxes, respectively. (<b>A</b>) tRNA<sup>Arg</sup>(UCU); (<b>B</b>) tRNA<sup>Asp</sup>(GUC); (<b>C</b>) tRNA<sup>Met</sup>(CAU-1); (<b>D</b>) tRNA<sup>Met</sup>(CAU-2); (<b>E</b>) tRNA<sup>Thr</sup>(CGU); (<b>F</b>) tRNA<sup>Pro</sup>(GGG); (<b>G</b>) tRNA<sup>Cys</sup>(GCA); (<b>H</b>) tRNA<sup>Tyr</sup>(GUA); (<b>I</b>) tRNA<sup>Thr</sup>(UGU-1); (<b>J</b>) tRNA<sup>Trp</sup>(CCA); (<b>K</b>) tRNA<sup>Lys</sup>(CUU); (<b>L</b>) tRNA<sup>Lys</sup>(UUU); (<b>M</b>) tRNA<sup>Pro</sup>(CGG).</p>
Full article ">Figure 5
<p>Summary of the tRNA introns identified in <span class="html-italic">S. tokodaii</span> strain7 [<a href="#B15-ijms-16-02187" class="html-bibr">15</a>]. Each label includes the tRNA species, the respective intron position relative to the 5' end of respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.</p>
Full article ">Figure 6
<p>Overall strategy used to clone cDNAs that represent actual <span class="html-italic">S. tokodaii</span> tRNAs [<a href="#B15-ijms-16-02187" class="html-bibr">15</a>].</p>
Full article ">Figure 7
<p>Exon-intron border regions of unspliced forms of tRNA<sup>Glu</sup>(UUC) (<b>A</b>); tRNA<sup>Glu</sup>(CUC) (<b>B</b>); and tRNA<sup>Leu</sup>(GAG) (<b>C</b>) of <span class="html-italic">S. tokodaii</span> strain7 [<a href="#B15-ijms-16-02187" class="html-bibr">15</a>]. These are examples of introns located at positions other than position “37/38”. In (<b>A</b>,<b>B</b>), only the D-arm region is shown; In (<b>C</b>), only the D-arm and anticodon arm regions are shown. The arrowheads indicate the exon-intron borders determined in our previous study [<a href="#B15-ijms-16-02187" class="html-bibr">15</a>]. The anticodon sequence of tRNA<sup>Leu</sup>(GAG) is boxed.</p>
Full article ">Figure 8
<p>The BHB-like structure in the unspliced form of tRNA<sup>Met</sup>(CAU-1) of <span class="html-italic">S. tokodaii</span> strain7 [<a href="#B15-ijms-16-02187" class="html-bibr">15</a>]. Only the anticodon arm and extra loop regions are shown. (<b>A</b>) The structure predicted by Marck and Grosjean [<a href="#B19-ijms-16-02187" class="html-bibr">19</a>]; (<b>B</b>) the structure inferred by <span class="html-italic">in vitro</span> cleavage of the intron with recombinant <span class="html-italic">S. tokodaii</span> EndA [<a href="#B15-ijms-16-02187" class="html-bibr">15</a>]. The exon-intron borders determined by the biochemical study [<a href="#B15-ijms-16-02187" class="html-bibr">15</a>] are indicated by arrowheads. Dashed lines in (<b>A</b>,<b>B</b>) show the cleavage sites predicted by Marck and Grosjean [<a href="#B19-ijms-16-02187" class="html-bibr">19</a>]. The anticodon sequences are boxed.</p>
Full article ">
2734 KiB  
Article
Transgenic Plants as Low-Cost Platform for Chemotherapeutic Drugs Screening
by Daniele Vergara, Stefania De Domenico, Michele Maffia, Gabriella Piro and Gian-Pietro Di Sansebastiano
Int. J. Mol. Sci. 2015, 16(1), 2174-2186; https://doi.org/10.3390/ijms16012174 - 20 Jan 2015
Cited by 9 | Viewed by 7169
Abstract
In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism [...] Read more.
In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects. Full article
(This article belongs to the Special Issue Plant Cell Compartmentation and Volume Control)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Fluorescent patterns of GFP-tagged marker proteins in transgenic Arabidopsis tissues in control conditions or treated with drugs targeting the cytoskeleton. (<b>A</b>) Normal fluorescent pattern of transgenes of (<b>A.1</b>) and (<b>A.2</b>) microtubules marker GFP-TUA6 in elongated petiole cells; (<b>A.3</b>) ER marker GFPKDEL in leaf epidermal cells; (<b>A.4</b>) Marker of the lytic vacuole, AleuGFP, in the lower face of the leaf; (<b>A.5</b>) AleuGFP, in the upper face of the leaf; and (<b>A.6</b>) Vacuolar marker characteristic of the direct transport ER-to-vacuole GFPChi; (<b>B</b>) Effect of treatment with 30 μM Paclitaxel on fluorescent pattern of (<b>B.1</b>) GFP-TUA6 in petiole cells; (<b>B.2</b>) GFPKDEL in leaf epidermis; (<b>B.3</b>) GFPChi in leaf epidermis; and (<b>C</b>) Effect of treatment with 200 μM Y-27632 on fluorescent pattern of (<b>C.1</b>) GFP-TUA6 in petiole cells showing no significant alteration; (<b>C.2</b>) GFP-TUA6 showing stronger effects; (<b>C.3</b>) GFPKDEL in leaf epidermis; (<b>C.4</b>) GFPChi faint fluorescence in leaf epidermis; and (<b>C.5</b>) GFPChi fluorescence secreted in the intercellular spaces of leaf mesophyll. GFP fluorescence and chlorophyll autofluorescence are shown in green and blue, respectively. Scale bar = 20 μm.</p>
Full article ">Figure 2
<p>(<b>A</b>) Fluorescent patterns of GFP-tagged marker proteins in transgenic Arabidopsis tissues in control conditions (<b>A.1</b>) GFP-TUA6 in petiole cells, (<b>A.2</b>) GFPKDEL, (<b>A.3</b>) AleuGFP, and (<b>A.4</b>) GFPChi in leaf epidermis or treated with drugs targeting the endomembranes; (<b>B</b>) Effect of treatment with Crizotinib at the concentration of 0.2 μM (<b>B.1</b>) or 20 μM (<b>B.2</b>) on fluorescent pattern of GFP-TUA6 in petiole cells; (<b>B3</b>) effect of 20 μM in leaf epidermis on GFPKDEL; (<b>B.4</b>) AleuGFP and (<b>B.5</b>) GFPChi distribution; and (<b>C</b>) Effect of treatment with Sorafenib at the concentration of (<b>C.1</b>) 2 μM on GFP-TUA6 in petiole cells or 1 μM on GFPKDEL (<b>C.2</b>) and AleuGFP (<b>C.3</b>) in leaf epidermis. GFP fluorescence and chlorophyll autofluorescence are shown in green and blue, respectively. Scale bar = 20 μm.</p>
Full article ">Figure 3
<p>Fluorescent patterns of GFPChi in transgenic Arabidopsis leaf epidermis treated with low doses of two combined drugs. Treatment with 0.2 μM Crizotinib had a moderate effect on GFPChi (<b>A</b>); but it was enhanced when combined with 10 μM Paclitaxel disturbing intermediate steps of its sorting (<b>B</b>) or causing mis-sorting to the apoplast when combined with 25 μM Y-27632 (<b>C</b>); combined treatment with 0.01 μM Sorafenib caused the increase of fluorescence in the central and small vacuoles (<b>D</b>). GFP fluorescence and chlorophyll autofluorescence are shown in green and blue, respectively. Scale bar = 20 μm.</p>
Full article ">
3874 KiB  
Article
Molecular Characterization of a New Wheat-Thinopyrum intermedium Translocation Line with Resistance to Powdery Mildew and Stripe Rust
by Haixian Zhan, Xiaojun Zhang, Guangrong Li, Zhihui Pan, Jin Hu, Xin Li, Linyi Qiao, Juqing Jia, Huijuan Guo, Zhijian Chang and Zujun Yang
Int. J. Mol. Sci. 2015, 16(1), 2162-2173; https://doi.org/10.3390/ijms16012162 - 20 Jan 2015
Cited by 23 | Viewed by 7990
Abstract
A new wheat-Thinopyrum translocation line CH13-21 was selected from the progenies derived from a cross between wheat-Th. intermedium partial amphiploid TAI7047 and wheat line Mianyang11. CH13-21 was characterized by using genomic in situ hybridization (GISH), multicolor-GISH (mc-GISH), multicolor-fluorescence in situ hybridization [...] Read more.
A new wheat-Thinopyrum translocation line CH13-21 was selected from the progenies derived from a cross between wheat-Th. intermedium partial amphiploid TAI7047 and wheat line Mianyang11. CH13-21 was characterized by using genomic in situ hybridization (GISH), multicolor-GISH (mc-GISH), multicolor-fluorescence in situ hybridization (mc-FISH) and chromosome-specific molecular markers. When inoculated with stripe rust and powdery mildew isolates, CH13-21 displayed novel resistance to powdery mildew and stripe rust which inherited from its Thinopyrum parent. The chromosomal counting analyses indicated that CH13-21 has 42 chromosomes, with normal bivalent pairing at metaphase I of meiosis. GISH probed by Th. intermedium genomic DNA showed that CH13-21 contained a pair of wheat-Th. intermedium translocated chromosomes. Sequential mc-FISH analyses probed by pSc119.2 and pAs1 clearly revealed that chromosome arm 6BS of CH13-21 was replaced by Thinopyrum chromatin in the translocation chromosome. The molecular markers analysis further confirmed that the introduced Th. intermedium chromatin in CH13-21 belonged to the long arm of homoeologous group 6 chromosome. Therefore, CH13-21 was a new T6BS.6Ai#1L compensating Robertsonian translocation line. It concludes that CH13-21 is a new genetic resource for wheat breeding programs providing novel variation for disease resistances. Full article
(This article belongs to the Special Issue Plant Molecular Biology)
Show Figures

Figure 1

Figure 1
<p>Response to stripe rust and powdery mildew of lines MY11 (<b>A</b>); CH13-21(<b>B</b>) and TAI7047 (<b>C</b>) on the leaves of adult plants.</p>
Full article ">Figure 2
<p>Genomic <span class="html-italic">in situ</span> hybridization (GISH) and sequential fluorescence <span class="html-italic">in situ</span> hybridization (FISH) patterns on the mitotic metaphase chromosomes of the common wheat line CH13-21. (<b>A</b>) GISH pattern with the labeled <span class="html-italic">Th. intermedium</span> genomic DNA as probe (green); (<b>B</b>) The multi-color (mc)-GISH pattern with labeled D-genomic DNA (red) and A-genomic DNA (green) as probes; (<b>C</b>) The mc-FISH pattern with the labeled pAs1 (red) and pSc119.2 (green) as probes. Arrows indicated the two translocated chromosomes, and scale bar showed 10 µm.</p>
Full article ">Figure 3
<p>PCR amplification using primers TNAC1726 (<b>A</b>); TNAC1702 (<b>B</b>) and TNAC1752 (<b>C</b>). The arrows indicates the <span class="html-italic">Th. intermedium</span> specific bands.</p>
Full article ">
1518 KiB  
Article
Unravelling Genes and Pathways Implicated in Working Memory of Schizophrenia in Han Chinese
by Hongyan Ren, Chengcheng Zhang, Chaohua Huang, Na Li, Mingli Li, Yinfei Li, Wei Deng, Xiaohong Ma, Bo Xiang, Qiang Wang and Tao Li
Int. J. Mol. Sci. 2015, 16(1), 2145-2161; https://doi.org/10.3390/ijms16012145 - 20 Jan 2015
Cited by 7 | Viewed by 6405
Abstract
Working memory deficit is the core neurocognitive disorder in schizophrenia patients. To identify the factors underlying working memory deficit in schizophrenia patients and to explore the implication of possible genes in the working memory using genome-wide association study (GWAS) of schizophrenia, computerized delay-matching-to-sample [...] Read more.
Working memory deficit is the core neurocognitive disorder in schizophrenia patients. To identify the factors underlying working memory deficit in schizophrenia patients and to explore the implication of possible genes in the working memory using genome-wide association study (GWAS) of schizophrenia, computerized delay-matching-to-sample (DMS) and whole genome genotyping data were obtained from 100 first-episode, treatment-nai?ve patients with schizophrenia and 140 healthy controls from the Mental Health Centre of the West China Hospital, Sichuan University. A composite score, delay-matching-to-sample total correct numbers (DMS-TC), was found to be significantly different between the patients and control. On associating quantitative DMS-TC with interactive variables of groups × genotype, one SNP (rs1411832), located downstream of YWHAZP5 in chromosome 10, was found to be associated with the working memory deficit in schizophrenia patients with lowest p-value (p = 2.02 × 10?7). ConsensusPathDB identified that genes with SNPs for which p values below the threshold of 5 × 10?5 were significantly enriched in GO:0007155 (cell adhesion, p < 0.001). This study indicates that working memory, as an endophenotype of schizophrenia, could improve the efficacy of GWAS in schizophrenia. However, further study is required to replicate the results from our study. Full article
(This article belongs to the Section Biochemistry)
Show Figures

Figure 1

Figure 1
<p>Multidimensional scaling plot of first two multidimensional scaling (MDS) components. Blue = control; Dark magenta = case.</p>
Full article ">Figure 2
<p>Q-Q plot of genome-wide association study (GWAS) on schizophrenia using DMS-TC (DMS-total correct numbers) as quantitative trait.</p>
Full article ">Figure 3
<p>Manhattan plots of genome-wide association of all SNPs with DMS-TC. SNPs were plotted on the x axis according to their position on each chromosome represented by difference color bars against association of DMS-TC on the y axis (shown as −log10P value).</p>
Full article ">
3184 KiB  
Article
Running Exercise Alleviates Pain and Promotes Cell Proliferation in a Rat Model of Intervertebral Disc Degeneration
by Shuo Luan, Qing Wan, Haijie Luo, Xiao Li, Songjian Ke, Caina Lin, Yuanyuan Wu, Shaoling Wu and Chao Ma
Int. J. Mol. Sci. 2015, 16(1), 2130-2144; https://doi.org/10.3390/ijms16012130 - 19 Jan 2015
Cited by 29 | Viewed by 8431
Abstract
Chronic low back pain accompanied by intervertebral disk degeneration is a common musculoskeletal disorder. Physical exercise, which is clinically recommended by international guidelines, has proven to be effective for degenerative disc disease (DDD) patients. However, the mechanism underlying the analgesic effects of physical [...] Read more.
Chronic low back pain accompanied by intervertebral disk degeneration is a common musculoskeletal disorder. Physical exercise, which is clinically recommended by international guidelines, has proven to be effective for degenerative disc disease (DDD) patients. However, the mechanism underlying the analgesic effects of physical exercise on DDD remains largely unclear. The results of the present study showed that mechanical withdrawal thresholds of bilateral hindpaw were significantly decreased beginning on day three after intradiscal complete Freund’s adjuvant (CFA) injection and daily running exercise remarkably reduced allodynia in the CFA exercise group beginning at day 28 compared to the spontaneous recovery group (controls). The hindpaw withdrawal thresholds of the exercise group returned nearly to baseline at the end of experiment, but severe pain persisted in the control group. Histological examinations performed on day 70 revealed that running exercise restored the degenerative discs and increased the cell densities of the annulus fibrosus (AF) and nucleus pulposus (NP). Furthermore, immunofluorescence labeling revealed significantly higher numbers of 5-bromo-2-deoxyuridine (BrdU)-positive cells in the exercise group on days 28, 42, 56 and 70, which indicated more rapid proliferation compared to the control at the corresponding time points. Taken together, these results suggest that running exercise might alleviate the mechanical allodynia induced by intradiscal CFA injection via disc repair and cell proliferation, which provides new evidence for future clinical use. Full article
(This article belongs to the Special Issue Molecular and Cellular Basis of Regeneration and Tissue Repair)
Show Figures

Figure 1

Figure 1
<p>Running exercise significantly attenuated the bilateral hindpaw mechanical allodynia induced by complete Freund’s adjuvant (CFA). The intradiscal CFA injection induced significant decreases in the mechanical withdrawal thresholds of the left (<b>A</b>) and right (<b>B</b>) hindpaws in response to von Frey filaments (<span class="html-italic">p</span> ˂ 0.01), however, no significant bilateral mechanical allodynia were observed in sham-operation groups (the sham exercise and the sham spontaneous recovery) compared to the baseline. For the CFA groups, the bilateral mechanical withdrawal thresholds were significantly increased in the exercise group compared to the spontaneous recovery group on day 28 (<span class="html-italic">p</span> ˂ 0.05) and days 42, 56 and 70 (<span class="html-italic">p</span> ˂ 0.01). The data are expressed as the mean ± SDs, <span class="html-italic">n</span> = 8 in each CFA groups, <span class="html-italic">n</span> = 5 in each sham-operation groups. Independent-samples <span class="html-italic">t</span> tests were used to examine the differences between the CFA running exercise subgroups <span class="html-italic">vs.</span> the CFA spontaneous recovery subgroups, and the sham exercise subgroups <span class="html-italic">vs.</span> the sham spontaneous recovery subgroups at the specific time points. We also used one-way analysis of variance (ANOVA) to analyze the within-group differences, and subsequent <span class="html-italic">post-hoc</span> tests were used to evaluate the differences between the specific time points and the baseline level in each group. <b>*</b> <span class="html-italic">p</span> ˂ 0.05, <b>**</b> <span class="html-italic">p</span> ˂ 0.01 compared to baseline; <sup>#</sup> <span class="html-italic">p</span> ˂ 0.05, <sup>##</sup> <span class="html-italic">p</span> ˂ 0.01 CFA exercise group compared to the CFA spontaneous recovery group at the corresponding time points.</p>
Full article ">Figure 1 Cont.
<p>Running exercise significantly attenuated the bilateral hindpaw mechanical allodynia induced by complete Freund’s adjuvant (CFA). The intradiscal CFA injection induced significant decreases in the mechanical withdrawal thresholds of the left (<b>A</b>) and right (<b>B</b>) hindpaws in response to von Frey filaments (<span class="html-italic">p</span> ˂ 0.01), however, no significant bilateral mechanical allodynia were observed in sham-operation groups (the sham exercise and the sham spontaneous recovery) compared to the baseline. For the CFA groups, the bilateral mechanical withdrawal thresholds were significantly increased in the exercise group compared to the spontaneous recovery group on day 28 (<span class="html-italic">p</span> ˂ 0.05) and days 42, 56 and 70 (<span class="html-italic">p</span> ˂ 0.01). The data are expressed as the mean ± SDs, <span class="html-italic">n</span> = 8 in each CFA groups, <span class="html-italic">n</span> = 5 in each sham-operation groups. Independent-samples <span class="html-italic">t</span> tests were used to examine the differences between the CFA running exercise subgroups <span class="html-italic">vs.</span> the CFA spontaneous recovery subgroups, and the sham exercise subgroups <span class="html-italic">vs.</span> the sham spontaneous recovery subgroups at the specific time points. We also used one-way analysis of variance (ANOVA) to analyze the within-group differences, and subsequent <span class="html-italic">post-hoc</span> tests were used to evaluate the differences between the specific time points and the baseline level in each group. <b>*</b> <span class="html-italic">p</span> ˂ 0.05, <b>**</b> <span class="html-italic">p</span> ˂ 0.01 compared to baseline; <sup>#</sup> <span class="html-italic">p</span> ˂ 0.05, <sup>##</sup> <span class="html-italic">p</span> ˂ 0.01 CFA exercise group compared to the CFA spontaneous recovery group at the corresponding time points.</p>
Full article ">Figure 2
<p>Photomicrographs of midsagittal sections of complete Freund’s adjuvant (CFA)-injected and sham-operation discs. (<b>A</b>,<b>B</b>) The discs of the CFA model rats exhibited dehydrated nucleus pulposus and blurred boundaries between the nucleus pulposus (NP) and anulus fibrosus (AF) areas at day 14 after CFA injection; (<b>C</b>,<b>D</b>) Intact NP and the clear distinction between NP and AF in discs of sham exercise groups at day 14 and day 70; (<b>E</b>,<b>F</b>) The gradual recovery processes were observed at day 42 and day 56 in the CFA exercise group; (<b>G</b>) Restored disc structures in the exercise group at day 70 after CFA injection; (<b>H</b>) Remarkable degenerative changes in the spontaneous recovery group at day 70 after CFA injection; (<b>I</b>,<b>J</b>) Quantitative results of NP and AF cell numbers in the CFA groups and sham-operation groups. The CFA exercise group revealed significant increases in both the NP (0.5-fold, <span class="html-italic">p</span> ˂ 0.05) and AF (1.4-fold, <span class="html-italic">p</span> ˂ 0.01) areas compared to the CFA spontaneous recovery group at day 70. The sham exercise group also showed significant increases of NP and AF compared to the baseline from day 42 (<span class="html-italic">p</span> ˂ 0.05) and maintained till the day 70. The sham spontaneous recovery group did not show obvious changes in NP or AF cell numbers throughout the experiment. The data are expressed as the means ± SDs, <span class="html-italic">n</span> = 8 in each CFA groups, <span class="html-italic">n</span> = 5 in each sham-operation groups. Independent-samples <span class="html-italic">t</span> tests were used to examine the differences between the CFA running exercise subgroups <span class="html-italic">vs.</span> the CFA spontaneous recovery subgroups, and the sham exercise subgroups <span class="html-italic">vs.</span> the sham spontaneous recovery subgroups at the specific time points. One-way analysis of variance (ANOVA) was used to analyze the within-group differences, and subsequent <span class="html-italic">post-hoc</span> tests were used to evaluate the differences between the specific time points and the baseline level in each group. <b>*</b> <span class="html-italic">p</span> ˂ 0.05, <b>**</b> <span class="html-italic">p</span> ˂ 0.01 compared to baseline; <sup>#</sup> <span class="html-italic">p</span> ˂ 0.05, <sup>##</sup> <span class="html-italic">p</span> ˂ 0.01 CFA exercise group compared to the CFA spontaneous recovery group at the corresponding time points. <sup>※</sup> <span class="html-italic">p</span> ˂ 0.05 sham exercise group compared to the sham spontaneous recovery group at the corresponding time points.</p>
Full article ">Figure 2 Cont.
<p>Photomicrographs of midsagittal sections of complete Freund’s adjuvant (CFA)-injected and sham-operation discs. (<b>A</b>,<b>B</b>) The discs of the CFA model rats exhibited dehydrated nucleus pulposus and blurred boundaries between the nucleus pulposus (NP) and anulus fibrosus (AF) areas at day 14 after CFA injection; (<b>C</b>,<b>D</b>) Intact NP and the clear distinction between NP and AF in discs of sham exercise groups at day 14 and day 70; (<b>E</b>,<b>F</b>) The gradual recovery processes were observed at day 42 and day 56 in the CFA exercise group; (<b>G</b>) Restored disc structures in the exercise group at day 70 after CFA injection; (<b>H</b>) Remarkable degenerative changes in the spontaneous recovery group at day 70 after CFA injection; (<b>I</b>,<b>J</b>) Quantitative results of NP and AF cell numbers in the CFA groups and sham-operation groups. The CFA exercise group revealed significant increases in both the NP (0.5-fold, <span class="html-italic">p</span> ˂ 0.05) and AF (1.4-fold, <span class="html-italic">p</span> ˂ 0.01) areas compared to the CFA spontaneous recovery group at day 70. The sham exercise group also showed significant increases of NP and AF compared to the baseline from day 42 (<span class="html-italic">p</span> ˂ 0.05) and maintained till the day 70. The sham spontaneous recovery group did not show obvious changes in NP or AF cell numbers throughout the experiment. The data are expressed as the means ± SDs, <span class="html-italic">n</span> = 8 in each CFA groups, <span class="html-italic">n</span> = 5 in each sham-operation groups. Independent-samples <span class="html-italic">t</span> tests were used to examine the differences between the CFA running exercise subgroups <span class="html-italic">vs.</span> the CFA spontaneous recovery subgroups, and the sham exercise subgroups <span class="html-italic">vs.</span> the sham spontaneous recovery subgroups at the specific time points. One-way analysis of variance (ANOVA) was used to analyze the within-group differences, and subsequent <span class="html-italic">post-hoc</span> tests were used to evaluate the differences between the specific time points and the baseline level in each group. <b>*</b> <span class="html-italic">p</span> ˂ 0.05, <b>**</b> <span class="html-italic">p</span> ˂ 0.01 compared to baseline; <sup>#</sup> <span class="html-italic">p</span> ˂ 0.05, <sup>##</sup> <span class="html-italic">p</span> ˂ 0.01 CFA exercise group compared to the CFA spontaneous recovery group at the corresponding time points. <sup>※</sup> <span class="html-italic">p</span> ˂ 0.05 sham exercise group compared to the sham spontaneous recovery group at the corresponding time points.</p>
Full article ">Figure 3
<p>Comparisons of the 5-bromo-2-deoxyuridine (BrdU)-positive cells in four different areas between the CFA exercise group and CFA spontaneous recovery group. (<b>A</b>) The peripheral epiphyseal cartilage (pEC) area of the exercise group at day 42; (<b>B</b>) The pEC area of the spontaneous recovery group at day 42; (<b>C</b>) the outer rings of the AF (AFo) area of the exercise group at day 42; (<b>D</b>) the AFo area of the spontaneous recovery group at day 42; and (<b>E</b>) Comparisons of the BrdU-positive cells between the CFA exercise group and the spontaneous recovery group. The BrdU-positive cells in the stem cell niche (SN) and pEC of the exercise group were significantly increased from the early time point of day 21 (<span class="html-italic">p</span> ˂ 0.05), peaked at day 28 (<span class="html-italic">p</span> ˂ 0.01) and were maintained at high levels until the end of the experiment compared to the spontaneous recovery group (<span class="html-italic">p</span> ˂ 0.05). The cell numbers in the AFo and the inner rings of the AF (AFi) areas of the exercise group were higher on days 28, 42, 56 and 70 (<span class="html-italic">p</span> ˂ 0.05) and peaked at the relatively later time point of day 42 (<span class="html-italic">p</span> ˂ 0.01). The data are expressed as the means ± SDs, <span class="html-italic">n</span> = 8 in each CFA groups. Independent-samples <span class="html-italic">t</span> tests were used to examine the differences between the CFA running exercise subgroups <span class="html-italic">vs.</span> the CFA spontaneous recovery subgroups at the specific time points. One-way analysis of variance (ANOVA) was used to analyze the within-group differences, and subsequent <span class="html-italic">post-hoc</span> tests were used to evaluate the differences between the specific time points and the baseline level in each group. <sup>#</sup> <span class="html-italic">p</span> ˂ 0.05, <sup>##</sup> <span class="html-italic">p</span> ˂ 0.01 compared to the CFA spontaneous recovery group at the corresponding time points.</p>
Full article ">Figure 3 Cont.
<p>Comparisons of the 5-bromo-2-deoxyuridine (BrdU)-positive cells in four different areas between the CFA exercise group and CFA spontaneous recovery group. (<b>A</b>) The peripheral epiphyseal cartilage (pEC) area of the exercise group at day 42; (<b>B</b>) The pEC area of the spontaneous recovery group at day 42; (<b>C</b>) the outer rings of the AF (AFo) area of the exercise group at day 42; (<b>D</b>) the AFo area of the spontaneous recovery group at day 42; and (<b>E</b>) Comparisons of the BrdU-positive cells between the CFA exercise group and the spontaneous recovery group. The BrdU-positive cells in the stem cell niche (SN) and pEC of the exercise group were significantly increased from the early time point of day 21 (<span class="html-italic">p</span> ˂ 0.05), peaked at day 28 (<span class="html-italic">p</span> ˂ 0.01) and were maintained at high levels until the end of the experiment compared to the spontaneous recovery group (<span class="html-italic">p</span> ˂ 0.05). The cell numbers in the AFo and the inner rings of the AF (AFi) areas of the exercise group were higher on days 28, 42, 56 and 70 (<span class="html-italic">p</span> ˂ 0.05) and peaked at the relatively later time point of day 42 (<span class="html-italic">p</span> ˂ 0.01). The data are expressed as the means ± SDs, <span class="html-italic">n</span> = 8 in each CFA groups. Independent-samples <span class="html-italic">t</span> tests were used to examine the differences between the CFA running exercise subgroups <span class="html-italic">vs.</span> the CFA spontaneous recovery subgroups at the specific time points. One-way analysis of variance (ANOVA) was used to analyze the within-group differences, and subsequent <span class="html-italic">post-hoc</span> tests were used to evaluate the differences between the specific time points and the baseline level in each group. <sup>#</sup> <span class="html-italic">p</span> ˂ 0.05, <sup>##</sup> <span class="html-italic">p</span> ˂ 0.01 compared to the CFA spontaneous recovery group at the corresponding time points.</p>
Full article ">Figure 4
<p>The correlations between the mechanical withdrawal thresholds and the cell counts of CFA exercise group. (<b>A</b>) The mechanical withdrawal thresholds were closely related to the increased cell numbers of nucleus pulposus (NP) (<span class="html-italic">r</span> = 0.98, <span class="html-italic">p</span> ˂ 0.01); (<b>B</b>) The tight correlation was also observed in annulus fibrosus (AF) (<span class="html-italic">r</span> = 0.96, <span class="html-italic">p</span> ˂ 0.01). The correlations between pain relief and cell counts of NP and AF were determined by Spearman rank correlation. <span class="html-italic">n</span> = 8 in each CFA exercise group.</p>
Full article ">Figure 5
<p>Time course of the experimental protocol ▲ indicates the time point of the complete Freund’s Adjuvant (CFA) injection. △ indicates the starting time of the daily running exercise and the BrdU application. ↑ indicates the time points of the hindpaw withdrawal mechanical threshold measurements. ⇧ indicates the time points of the immunofluorescence staining for the BrdU-positive cells. <b>*</b> indicates the time points of the histological examinations.</p>
Full article ">
1230 KiB  
Article
Prevention of Osteoporosis by Oral Administration of Phytate-Removed and Deamidated Soybean ?-Conglycinin
by Makoto Akao, Ryusuke Abe, Noriko Sato, Atsuko Hasegawa-Tanigome, Hitoshi Kumagai and Hitomi Kumagai
Int. J. Mol. Sci. 2015, 16(1), 2117-2129; https://doi.org/10.3390/ijms16012117 - 19 Jan 2015
Cited by 8 | Viewed by 7062
Abstract
Phytate-removed and deamidated soybean ?-conglycinin (PrDS) prepared by ion-exchange resins was supplemented to be 4% in the diet administered to ovariectomized rats to investigate its preventive effect on osteoporosis. The apparent calcium absorption rate decreased following ovariectomy and was not replenished by oral [...] Read more.
Phytate-removed and deamidated soybean ?-conglycinin (PrDS) prepared by ion-exchange resins was supplemented to be 4% in the diet administered to ovariectomized rats to investigate its preventive effect on osteoporosis. The apparent calcium absorption rate decreased following ovariectomy and was not replenished by oral administration of phytate-removed soybean ?-conglycinin (PrS) or casein. On the other hand, administration of PrDS restored the calcium absorption rate to the same level as the sham group. Markers of bone resorption, such as serum parathyroid hormone (PTH) and urinary deoxypyridinoline (DPD), increased, and the bone mineral density and breaking stress decreased following ovariectomy. However, PrDS supplementation suppressed the changes caused by the decrease in calcium absorption from the small intestine. Therefore, PrDS supplementation shows promise for the prevention of postmenopausal osteoporosis. Full article
(This article belongs to the Special Issue Bioactive Proteins and Peptides Derived from Food)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Apparent mineral absorption rate in sham-treated and ovariectomized rats fed each experimental diet for a week. Sham, non-ovariectomized and 20% egg albumin intake; control, ovariectomized and 20% egg albumin intake; phytate-removed soybean β-conglycinin (PrS), ovariectomized and 4% PrS + 16% egg albumin intake; Phytate-removed and deamidated soybean β-conglycinin (PrDS), ovariectomized and 4% PrDS + 16% egg albumin intake; casein, ovariectomized and 4% casein + 16% egg albumin intake. Each value shows the mean for six rats with the standard error (SE). Absorption values for the same mineral indicated by different letters are significantly different at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 2
<p>Serum parathyroid hormone (PTH) level in sham-treated and ovariectomized rats fed each experimental diet for eight weeks. Sham, non-ovariectomized and 20% egg albumin intake; control, ovariectomized and 20% egg albumin intake; PrS, ovariectomized and 4% PrS + 16% egg albumin intake; PrDS, ovariectomized and 4% PrDS + 16% egg albumin intake; casein, ovariectomized and 4% casein + 16% egg albumin intake. Values show the means for six rats with the SE. Values indicated by different letters are significantly different at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 3
<p>The urinary deoxypyridinoline (DPD) level in sham-treated and ovariectomized rats fed each experimental diet for eight weeks. Urinary DPD was standardized with urinary creatinine. Sham, non-ovariectomized and 20% egg albumin intake; control, ovariectomized and 20% egg albumin intake; PrS, ovariectomized and 4% PrS + 16% egg albumin intake; PrDS, ovariectomized and 4% PrDS + 16% egg albumin intake; casein, ovariectomized and 4% casein + 16% egg albumin intake. Values represent the means for six rats with the SE. Values indicated by different letters are significantly different at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 4
<p>Bone mineral density (BMD) at 4 mm from the endpoint of femurs in sham-treated and ovariectomized rats fed each experimental diet for eight weeks. (<b>A</b>) Cortical, (<b>B</b>) trabecular and (<b>C</b>) total BMD. Sham, non-ovariectomized and 20% egg albumin intake; control, ovariectomized and 20% egg albumin intake; PrS, ovariectomized and 4% PrS + 16% egg albumin intake; PrDS, ovariectomized and 4% PrDS + 16% egg albumin intake; casein, ovariectomized and 4% casein + 16% egg albumin intake. Values represent the means for six rats with the SE. Values indicated by different letters for the same BMD are significantly different at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 4 Cont.
<p>Bone mineral density (BMD) at 4 mm from the endpoint of femurs in sham-treated and ovariectomized rats fed each experimental diet for eight weeks. (<b>A</b>) Cortical, (<b>B</b>) trabecular and (<b>C</b>) total BMD. Sham, non-ovariectomized and 20% egg albumin intake; control, ovariectomized and 20% egg albumin intake; PrS, ovariectomized and 4% PrS + 16% egg albumin intake; PrDS, ovariectomized and 4% PrDS + 16% egg albumin intake; casein, ovariectomized and 4% casein + 16% egg albumin intake. Values represent the means for six rats with the SE. Values indicated by different letters for the same BMD are significantly different at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 5
<p>Bending stress of femurs in sham-treated and ovariectomized rats fed each experimental diet for eight weeks. Sham, non-ovariectomized and 20% egg albumin intake; control, ovariectomized and 20% egg albumin intake; PrS, ovariectomized and 4% PrS + 16% egg albumin intake; PrDS, ovariectomized and 4% PrDS + 16% egg albumin intake; casein, ovariectomized and 4% casein + 16% egg albumin intake. Values represent the means for six rats with the SE. Values indicated by different letters are significantly different at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">
1416 KiB  
Review
Antimicrobial Polymers with Metal Nanoparticles
by Humberto Palza
Int. J. Mol. Sci. 2015, 16(1), 2099-2116; https://doi.org/10.3390/ijms16012099 - 19 Jan 2015
Cited by 602 | Viewed by 31141
Abstract
Metals, such as copper and silver, can be extremely toxic to bacteria at exceptionally low concentrations. Because of this biocidal activity, metals have been widely used as antimicrobial agents in a multitude of applications related with agriculture, healthcare, and the industry in general. [...] Read more.
Metals, such as copper and silver, can be extremely toxic to bacteria at exceptionally low concentrations. Because of this biocidal activity, metals have been widely used as antimicrobial agents in a multitude of applications related with agriculture, healthcare, and the industry in general. Unlike other antimicrobial agents, metals are stable under conditions currently found in the industry allowing their use as additives. Today these metal based additives are found as: particles, ions absorbed/exchanged in different carriers, salts, hybrid structures, etc. One recent route to further extend the antimicrobial applications of these metals is by their incorporation as nanoparticles into polymer matrices. These polymer/metal nanocomposites can be prepared by several routes such as in situ synthesis of the nanoparticle within a hydrogel or direct addition of the metal nanofiller into a thermoplastic matrix. The objective of the present review is to show examples of polymer/metal composites designed to have antimicrobial activities, with a special focus on copper and silver metal nanoparticles and their mechanisms. Full article
(This article belongs to the Special Issue Antimicrobial Polymers)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>A summary of the main mechanisms behind the antimicrobial behavior of metal as separated according to the specific metal property responsible for this action: (<b>a</b>) reduction potential and (<b>b</b>) donor atom selectivity and/or speciation.</p>
Full article ">Figure 2
<p>A summary of the mechanisms associated with the antimicrobial behaviour of metal nanoparticles: (1) “Trojan-horse effect” due to endocytosis processes; (2) attachment to the membrane surface; (3) catalyzed radical formation; and (4) release of metal ions.</p>
Full article ">Figure 3
<p>Two main routes producing antimicrobial polymer/metal nanocomposites: (<b>a</b>) Polymer as reaction mediun for <span class="html-italic">in-situ</span> synthesis of nanoparticles; and (<b>b</b>) Polymer as a dispersion mediun of pre-synthesized nanoparticles.</p>
Full article ">Figure 4
<p>Mechanisms for the antimicrobial behavior of polymer/metal nanocomposites based on thermoplastic matrices: (<b>1</b>) adsorbtion of bacteria on the polymer surface triggering the diffusion of water through the polymer matrix due to the medium surrounded the bacteria; (<b>2</b>) water with dissolved oxygen reaches the surface of embedded metal nanoparticles allowing dissolution or corrosion processes and in this way metal ions are realized; (<b>3</b>) metal ions reach the composite surface damaging the bacteria membrane; (<b>4</b>) Afterward, metal ions can diffuse into the bacteria. The details of the specific mechanisms are explained in <a href="#ijms-16-02099-f001" class="html-fig">Figure 1</a>. Although this figure represents a polymer nanocomposite, it can be extrapolated to any polymer/metal materials with the biocide agent embedded in the matrix.</p>
Full article ">
2373 KiB  
Article
Insecticide-Mediated Up-Regulation of Cytochrome P450 Genes in the Red Flour Beetle (Tribolium castaneum)
by Xiao Liang, Da Xiao, Yanping He, Jianxiu Yao, Guonian Zhu and Kun Yan Zhu
Int. J. Mol. Sci. 2015, 16(1), 2078-2098; https://doi.org/10.3390/ijms16012078 - 19 Jan 2015
Cited by 53 | Viewed by 7330
Abstract
Some cytochrome P450 (CYP) genes are known for their rapid up-regulation in response to insecticide exposures in insects. To date, however, limited information is available with respect to the relationships among the insecticide type, insecticide concentration, exposure duration and the up-regulated CYP genes. [...] Read more.
Some cytochrome P450 (CYP) genes are known for their rapid up-regulation in response to insecticide exposures in insects. To date, however, limited information is available with respect to the relationships among the insecticide type, insecticide concentration, exposure duration and the up-regulated CYP genes. In this study, we examined the transcriptional response of eight selected CYP genes, including CYP4G7, CYP4Q4, CYP4BR3, CYP12H1, CYP6BK11, CYP9D4, CYP9Z5 and CYP345A1, to each of four insecticides in the red flour beetle, Tribolium castaneum. Reverse transcription quantitative PCR (RT-qPCR) revealed that CYP4G7 and CYP345A1 can be significantly up-regulated by cypermethrin (1.97- and 2.06-fold, respectively), permethrin (2.00- and 2.03-fold) and lambda-cyhalothrin (1.73- and 1.81-fold), whereas CYP4BR3 and CYP345A1 can be significantly up-regulated by imidacloprid (1.99- and 1.83-fold) when 20-day larvae were exposed to each of these insecticides at the concentration of LC20 for 24 h. Our studies also showed that similar levels of up-regulation can be achieved for CYP4G7, CYP4BR3 and CYP345A1 by cypermethrin, permethrin, lambda-cyhalothrin or imidacloprid with approximately one fourth of LC20 in 6 h. Our study demonstrated that up-regulation of these CYP genes was rapid and only required low concentrations of insecticides, and the up-regulation not only depended on the CYP genes but also the type of insecticides. Our results along with those from previous studies also indicated that there were no specific patterns for predicting the up-regulation of specific CYP gene families based on the insecticide classification. Full article
(This article belongs to the Section Molecular Toxicology)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Neighbor-joining phylogenetic trees of three CYP clans. (<b>A</b>) mitochondrial; (<b>B</b>) CYP4; and (<b>C</b>) CYP3. The trees were constructed by using MEGA 5 based on the full-length amino acid sequences deduced from the cDNA or genomic DNA sequences of <span class="html-italic">T. castaneum</span> (Tc), <span class="html-italic">D. melanogaster</span> (Dm), <span class="html-italic">A. gambiae</span> (Ag), <span class="html-italic">Musca domestica</span> (Md), <span class="html-italic">A. aegypti</span> (Aa), <span class="html-italic">C. pipiens pallens</span> (Cp), <span class="html-italic">C. quinquefasciatus</span> (Cq), <span class="html-italic">Blattella germanica</span> (Bg), <span class="html-italic">Helicoverpa armigera</span> (Ha), <span class="html-italic">Diabrotica virgifera virgifera</span> (Dv), <span class="html-italic">Manduca sexta</span> (Ms), <span class="html-italic">Bombyx mori</span> (Bm), <span class="html-italic">H. Zea</span> (Hz), <span class="html-italic">Plutella xylostella</span> (Px), <span class="html-italic">Chironomus tentans</span> (Ct), and <span class="html-italic">Apis mellifera</span> (Am). The accession number of each gene from NCBI is shown in bold at the end of the gene name. All nodes have significant bootstrap support based on 3000 replicates. The trees were constructed with cut-off value of 50%. The CYPs known to be implicated in insecticide resistance and up-regulation were indicated with a black triangle and a red dot, respectively, or both. In addition, sequence logos, which were predicted by WebLogo tool (<a href="http://weblogo.berkeley.edu/logo.cgi" target="_blank">http://weblogo.berkeley.edu/logo.cgi</a>), depicted the conservation of amino acid residues in CYP heme-binding motif of each clustered clade. The letter size is proportional to the degree of amino acid conservation. Eight CYPs selected for this study were boxed.</p>
Full article ">Figure 1 Cont.
<p>Neighbor-joining phylogenetic trees of three CYP clans. (<b>A</b>) mitochondrial; (<b>B</b>) CYP4; and (<b>C</b>) CYP3. The trees were constructed by using MEGA 5 based on the full-length amino acid sequences deduced from the cDNA or genomic DNA sequences of <span class="html-italic">T. castaneum</span> (Tc), <span class="html-italic">D. melanogaster</span> (Dm), <span class="html-italic">A. gambiae</span> (Ag), <span class="html-italic">Musca domestica</span> (Md), <span class="html-italic">A. aegypti</span> (Aa), <span class="html-italic">C. pipiens pallens</span> (Cp), <span class="html-italic">C. quinquefasciatus</span> (Cq), <span class="html-italic">Blattella germanica</span> (Bg), <span class="html-italic">Helicoverpa armigera</span> (Ha), <span class="html-italic">Diabrotica virgifera virgifera</span> (Dv), <span class="html-italic">Manduca sexta</span> (Ms), <span class="html-italic">Bombyx mori</span> (Bm), <span class="html-italic">H. Zea</span> (Hz), <span class="html-italic">Plutella xylostella</span> (Px), <span class="html-italic">Chironomus tentans</span> (Ct), and <span class="html-italic">Apis mellifera</span> (Am). The accession number of each gene from NCBI is shown in bold at the end of the gene name. All nodes have significant bootstrap support based on 3000 replicates. The trees were constructed with cut-off value of 50%. The CYPs known to be implicated in insecticide resistance and up-regulation were indicated with a black triangle and a red dot, respectively, or both. In addition, sequence logos, which were predicted by WebLogo tool (<a href="http://weblogo.berkeley.edu/logo.cgi" target="_blank">http://weblogo.berkeley.edu/logo.cgi</a>), depicted the conservation of amino acid residues in CYP heme-binding motif of each clustered clade. The letter size is proportional to the degree of amino acid conservation. Eight CYPs selected for this study were boxed.</p>
Full article ">Figure 2
<p>Stage-dependent (<b>A</b>) and tissue-dependent (<b>B</b>) expression patterns of eight selected CYP genes in <span class="html-italic">T. castaneum</span> (Georgia-1 strain). The expression profiles were evaluated by reverse transcription PCR (RT-PCR). The expression patterns of five different tissues, including foregut (FG), midgut (MG), hindgut (HG), Malpighian tubules (MT), fat bodies (FB) were derived from 20-day larvae, and <span class="html-italic">TcRPS3</span> was used as an internal reference gene.</p>
Full article ">Figure 3
<p>Up-regulation of CYP genes in <span class="html-italic">T. castaneum</span> after exposed to different insecticides. Dash lines represent relative transcript level of the control (larvae treated with the insecticide solvent only) as 1.0. The up-regulation fold was acquired by comparing the transcript levels of each CYP between the treated and the control insects. The CYP genes with a statistically significant up-regulation are marked with asterisks (Student’s <span class="html-italic">t</span> test, * <span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 4
<p>Cypermethrin concentration and time dependent up-regulation of <span class="html-italic">CYP4G7</span> and <span class="html-italic">CYP345A1</span> in 20-day larvae. Controls were normalized as 1.0, and the relative transcript levels of CYP genes were calculated based on their corresponding controls. (<b>A</b>) Cypermethrin concentration dependent up-regulation as measured at 2 and 0.5 μg/mL with the exposure time of 24 h. Different letters above the standard error bars indicate significant differences based on the one-way ANOVA followed by Tukey’s HSD multiple comparison test (<span class="html-italic">p</span> &lt; 0.05); (<b>B</b>) Time dependent up-regulation of <span class="html-italic">CYP4G7</span> by cypermethrin (0.5 μg/mL) as measured at 6, 12, 24 and 48 h; and (<b>C</b>) Time dependent up-regulation of <span class="html-italic">CYP345A1</span> by cypermethrin (0.5 μg/mL) as measured at 6, 12, 24 and 48 h. Dash lines represent relative transcript level of the control (larvae treated with the insecticide solvent only) as 1.0. Statistical analysis was conducted to compare the expression levels between the control and the insecticide-treated insects within the same time duration by using Student’s <span class="html-italic">t</span> test. Asterisk above the standard error bars indicates significant difference whereas NS indicates no significant difference.</p>
Full article ">Figure 5
<p>Insecticide concentration and time-dependent effect on the up-regulation of the CYP genes in 20-day larvae. The fold changes were also statistically compared between the two treatment combinations (<span class="html-italic">i.e</span>., 0.5 μg/mL for 6 h against 2 μg/mL for 24 h for cypermethrin and lambda-cyhalothrin, and 4 μg/mL for 6 h against 16 μg/mL for 24 h for permethrin and imidacloprid) by Student’s <span class="html-italic">t</span> test. An asterisk above the standard error bars indicates significant difference whereas NS indicates no significant difference.</p>
Full article ">
1252 KiB  
Article
Variation and Genetic Structure in Platanus mexicana (Platanaceae) along Riparian Altitudinal Gradient
by Dulce M. Galván-Hernández, J. Armando Lozada-García, Norma Flores-Estévez, Jorge Galindo-González and S. Mario Vázquez-Torres
Int. J. Mol. Sci. 2015, 16(1), 2066-2077; https://doi.org/10.3390/ijms16012066 - 19 Jan 2015
Cited by 5 | Viewed by 6275
Abstract
Platanus mexicana is a dominant arboreal species of riparian ecosystems. These ecosystems are associated with altitudinal gradients that can generate genetic differences in the species, especially in the extremes of the distribution. However, studies on the altitudinal effect on genetic variation to riparian [...] Read more.
Platanus mexicana is a dominant arboreal species of riparian ecosystems. These ecosystems are associated with altitudinal gradients that can generate genetic differences in the species, especially in the extremes of the distribution. However, studies on the altitudinal effect on genetic variation to riparian species are scarce. In Mexico, the population of P. mexicana along the Colipa River (Veracruz State) grows below its reported minimum altitude range, possibly the lowest where this tree grows. This suggests that altitude might be an important factor in population genetics differentiation. We examined the genetic variation and population structuring at four sites with different altitudes (70, 200, 600 and 1700 m a.s.l.) using ten inter-simple sequence repeats (ISSR) markers. The highest value for Shannon index and Nei’s gene diversity was obtained at 1700 m a.s.l. (He = 0.27, Ne = 1.47, I = 0.42) and polymorphism reached the top value at the middle altitude (% p = 88.57). Analysis of molecular variance (AMOVA) and STRUCTURE analysis indicated intrapopulation genetic differentiation. The arithmetic average (UPGMA) dendrogram identified 70 m a.s.l. as the most genetically distant site. The genetic structuring resulted from limited gene flow and genetic drift. This is the first report of genetic variation in populations of P. mexicana in Mexico. This research highlights its importance as a dominant species, and its ecological and evolutionary implications in altitudinal gradients of riparian ecosystems. Full article
(This article belongs to the Special Issue Plant Molecular Biology)
Show Figures

Figure 1

Figure 1
<p>UPGMA diagram based on Nei’s genetic distances for <span class="html-italic">P. mexicana</span> on the Colipa River, Veracruz, Mexico.</p>
Full article ">Figure 2
<p>Population structure based on ISSR variation among sites at <span class="html-italic">K</span> = 8, implemented in STRUCTURE, v. 2.3, where each line represents the proportional assignment of an individual to the clusters, represented by the different colors.</p>
Full article ">Figure 3
<p>Location of four sampled sites of <span class="html-italic">P. mexicana</span> in the Colipa River.</p>
Full article ">
3588 KiB  
Article
Isolation and Characterization of Six AP2/ERF Transcription Factor Genes in Chrysanthemum nankingense
by Chunyan Gao, Peiling Li, Aiping Song, Haibin Wang, Yinjie Wang, Liping Ren, Xiangyu Qi, Fadi Chen, Jiafu Jiang and Sumei Chen
Int. J. Mol. Sci. 2015, 16(1), 2052-2065; https://doi.org/10.3390/ijms16012052 - 19 Jan 2015
Cited by 24 | Viewed by 7233
Abstract
The AP2/ERF family of plant transcription factors (TFs) regulate a variety of developmental and physiological processes. Here, we report the isolation of six AP2/ERF TF family genes from Chrysanthemum nankingense. On the basis of sequence similarity, one of these belonged to the Ethylene [...] Read more.
The AP2/ERF family of plant transcription factors (TFs) regulate a variety of developmental and physiological processes. Here, we report the isolation of six AP2/ERF TF family genes from Chrysanthemum nankingense. On the basis of sequence similarity, one of these belonged to the Ethylene Responsive Factor (ERF) subfamily and the other five to the Dehydration Responsive Element Binding protein (DREB) subfamily. A transient expression experiment showed that all six AP2/ERF proteins localized to the nucleus. A yeast-one hybrid assay demonstrated that CnDREB1-1, 1-2 and 1-3 all function as transactivators, while CnERF1, CnDREB3-1 and 3-2 have no transcriptional activation ability. The transcription response of the six TFs in response to wounding, salinity and low temperature stress and treatment with abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) showed that CnERF1 was up-regulated by wounding and low temperature stress but suppressed by salinity stress. The transcription of CnDREB1-1, 1-2 and 1-3 was down-regulated by ABA and JA to varying degrees. CnDREB3-1 and 3-2 was moderately increased or decreased by wounding and SA treatment, suppressed by salinity stress and JA treatment, and enhanced by low temperature stress and ABA treatment. Full article
(This article belongs to the Special Issue Plant Molecular Biology)
Show Figures

Figure 1

Figure 1
<p>Deduced peptide sequences of the <span class="html-italic">CnAP2/ERF</span> transcription factor (TF) products and their phylogenetic relationship with <span class="html-italic">A. thaliana</span> homologs. (<b>a</b>) The deduced polypeptide sequences; residues shared by at least three of the six sequences are shown shaded, whereas those conserved across all six polypeptides are marked in dark grey. Asterisks indicate the conserved DNA-binding AP2/ERF domain, a double overline indicates the putative acidic domain and a black underline indicates the putative nuclear localization signal; (<b>b</b>) Phylogeny of the <span class="html-italic">CnAP2/ERF</span> TF products. Dots indicate likely homologs.</p>
Full article ">Figure 2
<p>Localization of transiently expressed <span class="html-italic">CnAP2/ERF</span> TF products in onion epidermal cells. The upper row shows the control <span class="html-italic">35S::GFP</span> signal, and each of the lower rows the signal from one of the <span class="html-italic">35S::CnAP2/ERF-GFP</span> transgenes. The left panel shows bright field images, the middle one green fluorescence signals detected at 488 nm and the right one the merged Green Fluorescent Protein (GFP) and bright field images. Bar: 50 μm.</p>
Full article ">Figure 3
<p>Transcriptional activation activity of the CnAP2/ERF TFs. (<b>a</b>) The structure of the <span class="html-italic">pGBKT7-CnAP2/ERF</span> plasmid; (<b>b</b>) the arrangement of yeast strains on the plate; (<b>c</b>) the growth of transformed yeast cells on SD/-His-Ade + 20 mg/mL X-α-gal medium. pCL1 and pGBKT7 are positive and negative controls, respectively.</p>
Full article ">Figure 4
<p>Differential transcript abundance of the <span class="html-italic">CnAP2/ERF</span> TFs in response to (<b>a</b>) salinity stress; (<b>b</b>) abscisic acid (ABA) treatment; (<b>c</b>) salicylic acid (SA) treatment; (<b>d</b>) jasmonic acid (JA) treatment; (<b>e</b>) low temperature stress; and (<b>f</b>) wounding. Green cells indicate suppressed and red ones enhanced levels of transcript abundance compared to the relevant control. Black cells represent no significant change of transcript abundance.</p>
Full article ">
1996 KiB  
Article
Efficient Synthesis of a Maghemite/Gold Hybrid Nanoparticle System as a Magnetic Carrier for the Transport of Platinum-Based Metallotherapeutics
by Pavel Štarha, David Smola, Jiří Tuček and Zdeněk Trávníček
Int. J. Mol. Sci. 2015, 16(1), 2034-2051; https://doi.org/10.3390/ijms16012034 - 16 Jan 2015
Cited by 17 | Viewed by 9273
Abstract
The preparation and thorough characterization of a hybrid magnetic carrier system for the possible transport of activated platinum-based anticancer drugs, as demonstrated for cisplatin (cis-[Pt(NH3)2Cl2], CDDP), are described. The final functionalized mag/Au–LA–CDDP* system consists of [...] Read more.
The preparation and thorough characterization of a hybrid magnetic carrier system for the possible transport of activated platinum-based anticancer drugs, as demonstrated for cisplatin (cis-[Pt(NH3)2Cl2], CDDP), are described. The final functionalized mag/Au–LA–CDDP* system consists of maghemite/gold nanoparticles (mag/Au) coated by lipoic acid (HLA; LA stands for deprotonated form of lipoic acid) and functionalized by activated cisplatin in the form of cis-[Pt(NH3)2(H2O)2]2+ (CDDP*). The relevant techniques (XPS, EDS, ICP-MS) proved the incorporation of the platinum-containing species on the surface of the studied hybrid system. HRTEM, TEM and SEM images showed the nanoparticles as spherical with an average size of 12 nm, while their superparamagnetic feature was proven by 57Fe Mössbauer spectroscopy. In the case of mag/Au, mag/Au–HLA and mag/Au–LA–CDDP*, weaker magnetic interactions among the Fe3+ centers of maghemite, as compared to maghemite nanoparticles (mag), were detected, which can be associated with the non-covalent coating of the maghemite surface by gold. The pH and time-dependent stability of the mag/Au–LA–CDDP* system in different media, represented by acetate (pH 5.0), phosphate (pH 7.0) and carbonate (pH 9.0) buffers and connected with the release of the platinum-containing species, showed the ability of CDDP* to be released from the functionalized nanosystem. Full article
(This article belongs to the Special Issue Bioactive Nanoparticles 2014)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The reaction pathway leading to the preparation of the mag/Au–LA–CDDP* nanoparticle system.</p>
Full article ">Figure 2
<p>HRTEM (<b>left</b>; 10 nm size bar), TEM (<b>top right</b>; 50 nm size bar) and SEM (<b>bottom right</b>; 500 nm size bar) images, as obtained for the studied functionalized mag/Au–LA–CDDP* nanoparticles.</p>
Full article ">Figure 3
<p>High-angle annular dark-field detector (HAADF)-STEM images of mag/Au nanoparticles (<b>a</b>–<b>d</b>; 9 nm size bars) showing iron (<b>b</b>), gold (<b>c</b>) and both iron and gold present in one nanoparticle (<b>d</b>).</p>
Full article ">Figure 4
<p>The results of the XPS spectroscopy of the mag/Au–LA–CDDP* nanosystems (red lines) and their comparison with mag/Au–HLA (black lines), given for the 0–750 eV region (<b>top</b>) with the details of the Pt4f/Au4f region at 67.5–90 eV (<b>bottom left</b>) and C1s region in the 280–295 eV range (<b>bottom right</b>).</p>
Full article ">Figure 5
<p>EDS spectra of the final functionalized mag/Au–LA–CDDP* nanoparticles and their synthetic intermediates, mag/Au–HLA, mag/Au and mag nanoparticles, with the assigned peaks of carbon, oxygen, iron and gold/platinum. Insets: detail of the gold/platinum region (depicted in the 9.25–10.00 keV range).</p>
Full article ">Figure 6
<p>Thermogravimetry (TG) and differential thermal analysis (DTA) results obtained for mag/Au–LA–CDDP* (red curves), mag/Au–HLA (dashed green curves) and mag/Au (dotted purple curves).</p>
Full article ">Figure 7
<p>The IR spectra of the mag/Au–HLA nanoparticles (green; the detail of the aliphatic C–H vibration region is given as an inset) and its comparison with the mag nanoparticles (mag; black) and free lipoic acid (HLA; red).</p>
Full article ">Figure 8
<p><sup>57</sup>Fe Mössbauer spectrum of the mag, mag/Au, mag/Au–HLA and mag/Au–LA–CDDP* nanoparticles, measured at 300 K and without an external magnetic field.</p>
Full article ">
1578 KiB  
Article
Designed Surface Residue Substitutions in [NiFe] Hydrogenase that Improve Electron Transfer Characteristics
by Isaac T. Yonemoto, Hamilton O. Smith and Philip D. Weyman
Int. J. Mol. Sci. 2015, 16(1), 2020-2033; https://doi.org/10.3390/ijms16012020 - 16 Jan 2015
Cited by 4 | Viewed by 7111
Abstract
Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii “Deep Ecotype” [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons [...] Read more.
Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii “Deep Ecotype” [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme. Full article
(This article belongs to the Special Issue Photosynthesis and Biological Hydrogen Production)
Show Figures

Figure 1

Figure 1
<p>Electrostatic models of <span class="html-italic">S. elongatus</span> ferredoxin PetF (<b>A</b>); <span class="html-italic">Spinacia oleracea</span> PFOR (PDB: 1FNB) (<b>B</b>); Clostridial [FeFe] hydrogenase (PDB: 1FEH) (<b>C</b>); and a structural model of the <span class="html-italic">A. macleodii</span> hydrogenase small subunit (<b>D</b>). Negatively charged residues found near the docking site are colored red in the structural model. Electrostatic models of <span class="html-italic">A. macleodii</span> hydrogenase variants G1 (<b>E</b>) and G2 (<b>F</b>) in the same orientation as (<b>D</b>). Circled areas highlight the region near the distal Fe-S cluster. All models were obtained from the PDB where codes are given, or generated by the threading modeler Phyre. Charges were modelled using the default vacuum electrostatic package in PyMOL. In all electrostatic models, red is negatively charged and blue is positively charged.</p>
Full article ">Figure 2
<p>Methyl viologen-mediated (<b>A</b>) and methyl viologen-free (dithionite only) (<b>B</b>)<span class="html-italic"> in vitro</span> hydrogen production assay from extracts of <span class="html-italic">E. coli</span> over-expressing the WT hydrogenase, G1 hydrogenase, and progressive substitutions to G2 hydrogenase (see <a href="#ijms-16-02020-t001" class="html-table">Table 1</a> for sequence identities). Activities are plotted on a log scale over a 10-fold and 100-fold ranges, respectively to compare fold improvements.</p>
Full article ">Figure 3
<p>Methyl viologen-mediated (<b>A</b>) and methyl viologen-free (dithionite only) (<b>B</b>)<span class="html-italic"> in vitro</span> hydrogen production assay from extracts of <span class="html-italic">E. coli</span> over-expressing the G1 hydrogenase and Δ15 and Δ22 truncations of the <span class="html-italic">C</span>-terminal tail.</p>
Full article ">Figure 4
<p>Methyl viologen-mediated<span class="html-italic"> in vitro</span> hydrogen production assay (<b>A</b>) from tandem immobilized metal affinity chromatography (IMAC)/streptactin-purified G1 and G2 hydrogenases, and their respective ferredoxin fusions. <span class="html-italic">In vitro</span> H<sub>2</sub> production of the same hydrogenases in the methyl viologen-free assay (dithionite only) (<b>B</b>) and expressed as the ratio of methyl viologen-free activity to activity in assays containing methyl viologen (<b>C</b>), plotted on a log scale over an 100-fold range to compare fold-changes<span class="html-italic">.</span> Sypro-ruby stained gel (<b>D</b>) and Anti-HynL western blot (<b>E</b>) of the same constructs from the same protein preparations presented in (<b>A</b>,<b>B</b>).</p>
Full article ">
1499 KiB  
Article
Meshless Method with Operator Splitting Technique for Transient Nonlinear Bioheat Transfer in Two-Dimensional Skin Tissues
by Ze-Wei Zhang, Hui Wang and Qing-Hua Qin
Int. J. Mol. Sci. 2015, 16(1), 2001-2019; https://doi.org/10.3390/ijms16012001 - 16 Jan 2015
Cited by 10 | Viewed by 6577
Abstract
A meshless numerical scheme combining the operator splitting method (OSM), the radial basis function (RBF) interpolation, and the method of fundamental solutions (MFS) is developed for solving transient nonlinear bioheat problems in two-dimensional (2D) skin tissues. In the numerical scheme, the nonlinearity caused [...] Read more.
A meshless numerical scheme combining the operator splitting method (OSM), the radial basis function (RBF) interpolation, and the method of fundamental solutions (MFS) is developed for solving transient nonlinear bioheat problems in two-dimensional (2D) skin tissues. In the numerical scheme, the nonlinearity caused by linear and exponential relationships of temperature-dependent blood perfusion rate (TDBPR) is taken into consideration. In the analysis, the OSM is used first to separate the Laplacian operator and the nonlinear source term, and then the second-order time-stepping schemes are employed for approximating two splitting operators to convert the original governing equation into a linear nonhomogeneous Helmholtz-type governing equation (NHGE) at each time step. Subsequently, the RBF interpolation and the MFS involving the fundamental solution of the Laplace equation are respectively employed to obtain approximated particular and homogeneous solutions of the nonhomogeneous Helmholtz-type governing equation. Finally, the full fields consisting of the particular and homogeneous solutions are enforced to fit the NHGE at interpolation points and the boundary conditions at boundary collocations for determining unknowns at each time step. The proposed method is verified by comparison of other methods. Furthermore, the sensitivity of the coefficients in the cases of a linear and an exponential relationship of TDBPR is investigated to reveal their bioheat effect on the skin tissue. Full article
(This article belongs to the Special Issue Advances in Anisotropic and Smart Materials)
Show Figures

Figure 1

Figure 1
<p>Finite element mesh used in ANSYS.</p>
Full article ">Figure 2
<p>Collocation scheme with 63 interpolation points and 32 boundary collocations.</p>
Full article ">Figure 3
<p>Results for the linear case of blood perfusion rate (The arrow is indicator of zoom in image of the three overlap points in temperature curves. So that the reader can view the curves in details clearly).</p>
Full article ">Figure 4
<p>Results for the exponential case of blood perfusion rate (The arrow is indicator of zoom in image of the four overlap points in the temperature curves. So that the reader can view the curves in details clearly).</p>
Full article ">Figure 5
<p>Variation of temperature with time for the linear case of blood perfusion rate.</p>
Full article ">Figure 6
<p>Temperature variation <span class="html-italic">vs.</span> time along <span class="html-italic">x</span>-axis for the linear-form blood perfusion rate.</p>
Full article ">Figure 7
<p>Temperature variation <span class="html-italic">vs.</span> time along <span class="html-italic">x</span>-axis for the exponent-form blood perfusion rate.</p>
Full article ">Figure 8
<p>Sensitivity of temperature to constant <span class="html-italic">a</span><sub>1</sub> in the linear case of blood perfusion rate.</p>
Full article ">Figure 9
<p>Sensitivity of temperature to constant <span class="html-italic">a</span><sub>2</sub> in the linear case of blood perfusion rate.</p>
Full article ">Figure 10
<p>Sensitivity of temperature to constant <span class="html-italic">a</span><sub>1</sub> in the exponential case of blood perfusion rate.</p>
Full article ">Figure 11
<p>Sensitivity to constant <span class="html-italic">a</span><sub>2</sub> in the exponential case of blood perfusion rate.</p>
Full article ">Figure 12
<p>Two-dimensional skin model.</p>
Full article ">
1131 KiB  
Review
Medullary Thymic Epithelial Cells and Central Tolerance in Autoimmune Hepatitis Development: Novel Perspective from a New Mouse Model
by Konstantina Alexandropoulos, Anthony J. Bonito, Erica G. Weinstein and Olivier Herbin
Int. J. Mol. Sci. 2015, 16(1), 1980-2000; https://doi.org/10.3390/ijms16011980 - 16 Jan 2015
Cited by 12 | Viewed by 14898
Abstract
Autoimmune hepatitis (AIH) is an immune-mediated disorder that affects the liver parenchyma. Diagnosis usually occurs at the later stages of the disease, complicating efforts towards understanding the causes of disease development. While animal models are useful for studying the etiology of autoimmune disorders, [...] Read more.
Autoimmune hepatitis (AIH) is an immune-mediated disorder that affects the liver parenchyma. Diagnosis usually occurs at the later stages of the disease, complicating efforts towards understanding the causes of disease development. While animal models are useful for studying the etiology of autoimmune disorders, most of the existing animal models of AIH do not recapitulate the chronic course of the human condition. In addition, approaches to mimic AIH-associated liver inflammation have instead led to liver tolerance, consistent with the high tolerogenic capacity of the liver. Recently, we described a new mouse model that exhibited spontaneous and chronic liver inflammation that recapitulated the known histopathological and immunological parameters of AIH. The approach involved liver-extrinsic genetic engineering that interfered with the induction of T-cell tolerance in the thymus, the very process thought to inhibit AIH induction by liver-specific expression of exogenous antigens. The mutation led to depletion of specialized thymic epithelial cells that present self-antigens and eliminate autoreactive T-cells before they exit the thymus. Based on our findings, which are summarized below, we believe that this mouse model represents a relevant experimental tool towards elucidating the cellular and molecular aspects of AIH development and developing novel therapeutic strategies for treating this disease. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Human Liver Diseases)
Show Figures

Figure 1

Figure 1
<p>Schematic representation of T-cell selection and tolerance induction in the thymus. Bone marrow progenitors enter the thymus at the corticomedullary junction (CMJ) and migrate towards the cortex, where they differentiate into immature thymocytes lacking expression of the CD4 and CD8 coreceptors (double-negative 1-4, DN1-4). Upregulation of CD4 and CD8 gives rise to CD4<sup>+</sup>CD8<sup>+</sup> double-positive (DP) thymocytes whose T-cell receptor binds to self-antigens presented by cortical thymic epithelial cells (cTEC). A fraction of DP thymocytes are positively selected and differentiate into CD4<sup>+</sup> or CD8<sup>+</sup>single-positive (SP) T-cells. These migrate to the medulla, where they bind to tissue-restricted antigens (TRA) presented by medullary TECs (mTEC) and whose expression is regulated by Aire [<a href="#B30-ijms-16-01980" class="html-bibr">30</a>,<a href="#B31-ijms-16-01980" class="html-bibr">31</a>]. Autoreactive T-cells that bind to TRAs with high affinity are negatively selected. SP T-cells with weak affinity for TRAs are allowed to exit the thymus, whereas T-cells with intermediate affinity for TRAs become regulatory T-cells (Treg). mTEC depletion in Traf6∆TEC mice leads to the production of autoreactive T-cells, impaired production of Tregs and peripheral autoimmunity.</p>
Full article ">Figure 2
<p>Working model of AIH induction in Traf6∆TEC mice. mTEC depletion in the thymus of Traf6∆TEC mice results in the production of autoreactive CD4<sup>+</sup> T-cells, which home to the liver. In the liver, autoreactive CD4<sup>+</sup> T-cells aberrantly recognize liver-specific antigens presented by APCs, leading to T-cell activation. Activated T-cells differentiate into Th1 or Th2 effector cells, secreting IFN-γ or IL-4, respectively. IFN-γ mediates the activation of cytotoxic CD8<sup>+</sup> T-cells, whereas IL-4 induces the formation of plasma cells and autoantibody production, including ANAs and anti-SLA, leading to hepatocyte destruction and liver injury. Liver antigen-specific Tregs produced locally in response to inflammation counteract effector T-cell-mediated inflammation, leading to chronic AIH.</p>
Full article ">
883 KiB  
Review
Ligand-Induced Dynamics of Neurotrophin Receptors Investigated by Single-Molecule Imaging Approaches
by Laura Marchetti, Stefano Luin, Fulvio Bonsignore, Teresa De Nadai, Fabio Beltram and Antonino Cattaneo
Int. J. Mol. Sci. 2015, 16(1), 1949-1979; https://doi.org/10.3390/ijms16011949 - 16 Jan 2015
Cited by 15 | Viewed by 8376
Abstract
Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin [...] Read more.
Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells. Full article
Show Figures

Figure 1

Figure 1
<p>Schematic picture of tropomyosin receptor kinases (Trks) and the p75 neurotrophin receptors (p75NTR). (<b>A</b>) Structure of the two receptors: The intracellular (on <b>top</b>) and extracellular (on <b>bottom</b>) domains are highlighted. The following abbreviations are used: CRD (cystein-rich domain); LRR (leucine-rich domain); IgL-D (immunoglobulin-like domain); TKD (tyrosine-kinase domain); CD (chopper domain); DD (death domain); (<b>B</b>) Modified residues of the Trks (on <b>top</b>) and of p75NTR (on <b>bottom</b>) receptors. The following abbreviations are used: TM (transmembrane domain); JM (juxta-membrane domain); <span class="html-italic">N</span> (<span class="html-italic">N</span>-terminus); <span class="html-italic">C</span> (<span class="html-italic">C</span>-terminus). The following symbols are used: Grey ↓ (N- or O-glycosylation sites); green ↓ (ATP-binding site); red ↓ (site of covalent homo-dimerization due to the disulfide bond formed by Cys257 [<a href="#B32-ijms-16-01949" class="html-bibr">32</a>,<a href="#B33-ijms-16-01949" class="html-bibr">33</a>]). U (ubiquitination-related lysine residues, as derived from studies mainly performed on the TrkA receptor [<a href="#B34-ijms-16-01949" class="html-bibr">34</a>,<a href="#B35-ijms-16-01949" class="html-bibr">35</a>,<a href="#B36-ijms-16-01949" class="html-bibr">36</a>,<a href="#B37-ijms-16-01949" class="html-bibr">37</a>]); Y (phosphorylated tyrosine residues, their numeration and function is described in <a href="#ijms-16-01949-f002" class="html-fig">Figure 2</a>); P (palmitoylated Cys residue [<a href="#B38-ijms-16-01949" class="html-bibr">38</a>]).</p>
Full article ">Figure 2
<p>Schematic picture of four different NTs binding to ACP-TrkA. The ACP-TrkA construct and the four ligands investigated for receptor binding [<a href="#B104-ijms-16-01949" class="html-bibr">104</a>] are schematically depicted. NGF, NGF R100E mutant (mutNGF, related to HSANV disease [<a href="#B132-ijms-16-01949" class="html-bibr">132</a>]), proNGF and NT-3 all bind to the extracellular domain of TrkA receptor but with different affinity, as quantified by the <span class="html-italic">K<sub>d</sub></span> (dissociation constant) values (see the color-coded arrowheads referring to the corresponding <span class="html-italic">K<sub>d</sub></span> values, which are taken from: <b><span class="html-italic"><sup>≠</sup></span></b> [<a href="#B133-ijms-16-01949" class="html-bibr">133</a>]; <b>*</b> [<a href="#B134-ijms-16-01949" class="html-bibr">134</a>]; <b><sup>†</sup></b> [<a href="#B135-ijms-16-01949" class="html-bibr">135</a>]). The evoked physiological responses are also different among the four ligands and are summarized at the <span class="html-italic">C</span>-terminus of the receptor, highlighted by arrowheads with the same color-code. Intracellular effectors recruited at phosphorylated tyrosine residues and leading to the activation of the MAP kinase, the Akt and PLCγ signaling pathways, upon TrkA-NT binding are also schematically depicted. The numbering of tyrosine residues refers to the rat TrkA cDNA sequence. Note that while Y499 and 794 only have recruitment function, Y683/4 constitute with Y679 (not depicted) the activation loop of tyrosine kinase activity. The figure has been adapted from [<a href="#B104-ijms-16-01949" class="html-bibr">104</a>].</p>
Full article ">
3366 KiB  
Article
BRD4 Inhibitor Inhibits Colorectal Cancer Growth and Metastasis
by Yuan Hu, Jieqiong Zhou, Fei Ye, Huabao Xiong, Liang Peng, Zihan Zheng, Feihong Xu, Miao Cui, Chengguo Wei, Xinying Wang, Zhongqiu Wang, Hongfa Zhu, Peng Lee, Mingming Zhou, Bo Jiang and David Y. Zhang
Int. J. Mol. Sci. 2015, 16(1), 1928-1948; https://doi.org/10.3390/ijms16011928 - 16 Jan 2015
Cited by 83 | Viewed by 11607
Abstract
Post-translational modifications have been identified to be of great importance in cancers and lysine acetylation, which can attract the multifunctional transcription factor BRD4, has been identified as a potential therapeutic target. In this paper, we identify that BRD4 has an important role in [...] Read more.
Post-translational modifications have been identified to be of great importance in cancers and lysine acetylation, which can attract the multifunctional transcription factor BRD4, has been identified as a potential therapeutic target. In this paper, we identify that BRD4 has an important role in colorectal cancer; and that its inhibition substantially wipes out tumor cells. Treatment with inhibitor MS417 potently affects cancer cells, although such effects were not always outright necrosis or apoptosis. We report that BRD4 inhibition also limits distal metastasis by regulating several key proteins in the progression of epithelial-to-mesenchymal transition (EMT). This effect of BRD4 inhibitor is demonstrated via liver metastasis in animal model as well as migration and invasion experiments in vitro. Together, our results demonstrate a new application of BRD4 inhibitor that may be of clinical use by virtue of its ability to limit metastasis while also being tumorcidal. Full article
(This article belongs to the Section Biochemistry)
Show Figures

Figure 1

Figure 1
<p>BRD4 is highly expressed in colorectal cancer (CRC). (<b>A</b>) mRNA expression levels of BRD2, BRD4 short isoform and BRD4 long isoform in normal colon epithelial cell lines and colon cancer cell lines; (<b>B</b>) Protein levels of BRD4 in normal and colon cancer human tissues (tumor and healthy control); (<b>C</b>) Protein levels of BRD4 according to different ages; (<b>D</b>) Protein levels of BRD4 according to the stage of CRC (per AJCC guidelines). BRD4S: BRD4 short isoform; BRD4L: BRD4 long isoform; BRD4C: Commercial BRD4 primer as positive control; CRC: colorectal cancer.</p>
Full article ">Figure 2
<p>BRD4 inhibition attenuates proliferation, migration and invasion<span class="html-italic"> in vitro</span>. (<b>A</b>) 72 h survival rate curve of HT29, HCT8, HCT116, SW480 and SW620 treated with vehicle (DMSO) or MS417 (1, 3, 6, 9, 12, 24, 30 µM), measured by MTT; (<b>B</b>) IC<sub>50</sub> of HT29, HCT8, HCT116, SW480 and SW620 following vehicle/MS417 for 24, 48 and 72 h; (<b>C</b>,<b>D</b>) macroscopic and microscopic images and quantification of colonies formed by HT29 or SW620 cell lines treated with MS417 (1 µM); (<b>E</b>,<b>F</b>) Cell migration and invasion of HT29 or SW620 cell lines were inhibited by MS417 (1 µM). <span class="html-italic">n</span> = 3 repeats with similar results. <b>**</b> <span class="html-italic">p</span> &lt; 0.01; <b>***</b> <span class="html-italic">p</span> &lt; 0.001. Values are depicted as Mean ± SEM.</p>
Full article ">Figure 2 Cont.
<p>BRD4 inhibition attenuates proliferation, migration and invasion<span class="html-italic"> in vitro</span>. (<b>A</b>) 72 h survival rate curve of HT29, HCT8, HCT116, SW480 and SW620 treated with vehicle (DMSO) or MS417 (1, 3, 6, 9, 12, 24, 30 µM), measured by MTT; (<b>B</b>) IC<sub>50</sub> of HT29, HCT8, HCT116, SW480 and SW620 following vehicle/MS417 for 24, 48 and 72 h; (<b>C</b>,<b>D</b>) macroscopic and microscopic images and quantification of colonies formed by HT29 or SW620 cell lines treated with MS417 (1 µM); (<b>E</b>,<b>F</b>) Cell migration and invasion of HT29 or SW620 cell lines were inhibited by MS417 (1 µM). <span class="html-italic">n</span> = 3 repeats with similar results. <b>**</b> <span class="html-italic">p</span> &lt; 0.01; <b>***</b> <span class="html-italic">p</span> &lt; 0.001. Values are depicted as Mean ± SEM.</p>
Full article ">Figure 3
<p>BRD4 inhibition impacts transcriptional programs that control cell proliferation and EMT. (<b>A</b>) Apoptosis of HT29 and SW620 cell lines treated with vehicle (DMSO), MS417 (1 µM) or MS417 (12 µM); (<b>B</b>) Protein levels of candidate apoptosis-related factors following vehicle/MS417 treatment (1 and 12 µM) for 48 h; (<b>C</b>) Protein levels of E-cadherin and Vimentin in HT29 or SW620 cell lines treated with vehicle or MS417 (1 and 12 µM). The expression level of each protein was normalized against GAPDH. <b>*</b> <span class="html-italic">p</span> &lt; 0.01; <b>**</b> <span class="html-italic">p</span> &lt; 0.001; n.s. not significant.</p>
Full article ">Figure 3 Cont.
<p>BRD4 inhibition impacts transcriptional programs that control cell proliferation and EMT. (<b>A</b>) Apoptosis of HT29 and SW620 cell lines treated with vehicle (DMSO), MS417 (1 µM) or MS417 (12 µM); (<b>B</b>) Protein levels of candidate apoptosis-related factors following vehicle/MS417 treatment (1 and 12 µM) for 48 h; (<b>C</b>) Protein levels of E-cadherin and Vimentin in HT29 or SW620 cell lines treated with vehicle or MS417 (1 and 12 µM). The expression level of each protein was normalized against GAPDH. <b>*</b> <span class="html-italic">p</span> &lt; 0.01; <b>**</b> <span class="html-italic">p</span> &lt; 0.001; n.s. not significant.</p>
Full article ">Figure 4
<p>BRD4 inhibition impairs CRC tumor growth<span class="html-italic"> in vivo</span>. (<b>A</b>) Macroscopic images of resected tumors at the conclusion of the experiment; (<b>B</b>) average weight of resected tumors; (<b>C</b>) average tumor volume of mice injected with either vehicle (DMSO) or MS417 (20 mg/kg, <span class="html-italic">n</span> = 4/treatment); (<b>D</b>) average mice weights with every 2-day injection of vehicle/MS417 over a time course of 3 weeks. <b>*</b>, <span class="html-italic">p</span> &lt; 0.05; <b>**</b>, <span class="html-italic">p</span> &lt; 0.05; <b>***</b>, <span class="html-italic">p</span> &lt; 0.001. Values are depicted as Mean ± SEM.</p>
Full article ">Figure 5
<p>BRD4 inhibition suppresses CRC liver metastasis<span class="html-italic"> in vivo</span>. (<b>A</b>,<b>B</b>) Macroscopic images of resected livers at the conclusion of the experiment in mice injected by HT29 or SW620 cell lines and then treated by vehicle/MS417 (20 mg/kg, <span class="html-italic">n</span> = 5/treatment); (<b>C</b>,<b>D</b>) Representative microscopic H&amp;E images of livers. Metastatic focus is circled. <b>***</b> <span class="html-italic">p</span> &lt; 0.001. Values are depicted as Mean ± SEM.</p>
Full article ">Figure 5 Cont.
<p>BRD4 inhibition suppresses CRC liver metastasis<span class="html-italic"> in vivo</span>. (<b>A</b>,<b>B</b>) Macroscopic images of resected livers at the conclusion of the experiment in mice injected by HT29 or SW620 cell lines and then treated by vehicle/MS417 (20 mg/kg, <span class="html-italic">n</span> = 5/treatment); (<b>C</b>,<b>D</b>) Representative microscopic H&amp;E images of livers. Metastatic focus is circled. <b>***</b> <span class="html-italic">p</span> &lt; 0.001. Values are depicted as Mean ± SEM.</p>
Full article ">
6414 KiB  
Article
Melatonin Stimulates Dendrite Formation and Complexity in the Hilar Zone of the Rat Hippocampus: Participation of the Ca++/Calmodulin Complex
by Aline Domínguez-Alonso, Marcela Valdés-Tovar, Héctor Solís-Chagoyán and Gloria Benítez-King
Int. J. Mol. Sci. 2015, 16(1), 1907-1927; https://doi.org/10.3390/ijms16011907 - 16 Jan 2015
Cited by 45 | Viewed by 7038
Abstract
Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+ [...] Read more.
Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+/Calmodulin (CaM)-dependent kinase II (CaMKII) by CaM. Previous work showed that MEL increased the synthesis and translocation of CaM, suggesting that MEL activates CaM-dependent enzymes by this pathway. In this work we investigated whether MEL stimulates dendrite formation by CaMKII activation in organotypic cultures from adult rat hippocampus. We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices. Also, PKC inhibition abolished dendritogenesis, while luzindole, an antagonist of MEL receptors (MT1/2), partially blocked the effects of MEL. Moreover, autophosphorylation of CaMKII and PKC was increased in presence of MEL, as well as phosphorylation of ERK1/2. Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction. Dendritogenesis elicited by MEL also required PKC activation, and signaling through MT1/2 receptors was partially involved. Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII. Full article
(This article belongs to the Special Issue Advances in the Research of Melatonin 2014)
Show Figures

Figure 1

Figure 1
<p>Effect of Ca<sup>2+</sup>/CaM-Kinase II on dendrite formation elicited by Melatonin. Participation of CaMKII on dendrite formation elicited by MEL was evaluated by specific inhibition of its activity with KN-62. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal<sup>®</sup> (GIBCO by Life Technologies, Grand Island, NY, USA) media for 7 days. Then they were incubated for 6 h with either the vehicle (<b>A</b>); 100 nM MEL (<b>B</b>); or pre-incubated with 10 µM KN-62 (<b>C</b>,<b>D</b>) followed by 6 h incubation with the vehicle (<b>C</b>) or 100 nM MEL (<b>D</b>). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Afterwards, slices were incubated with a secondary antibody coupled to biotin-avidin-peroxidase. Images were acquired with a digital camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.</p>
Full article ">Figure 2
<p>Effect of Protein Kinase C on dendrite formation elicited by Melatonin. Participation of PKC on dendrite formation elicited by MEL was evaluated by specific inhibition of its activity with bisindolylmaleimide. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal<sup>®</sup> media for 7 days. Then they were incubated for 6 h with either the vehicle (<b>A</b>); 100 nM MEL (<b>B</b>); or pre-incubated with 5 µM of bisindolylmaleimide (<b>C</b>,<b>D</b>) followed by 6 h incubation with the vehicle (<b>C</b>) or 100 nM MEL (<b>D</b>). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Afterwards, slices were incubated with a secondary antibody coupled to biotin-avidin-peroxidase. Images were acquired with a digital camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.</p>
Full article ">Figure 3
<p>Morphometric analysis of dendritogenesis elicited by Melatonin: participation of Ca<sup>2+</sup>/CaM Kinase II and Protein Kinase C. The three stages of dendrite formation were evaluated by measuring the number and length of primary dendrites, as well as the number and length of secondary and tertiary dendrites. Hippocampal slices were incubated for 6 h with the following treatments: vehicle (VEH), 100 nM melatonin (MEL), pre- incubation of 15 min with 5 µM of the PKC inhibitor, bisindolylmaleimide (BIS) followed by the vehicle (BIS + VEH) or melatonin (BIS + MEL) for 6 h, or pre- incubation of 15 min with 10 µM of the CaMKII inhibitor, the KN-62 compound (KN-62) followed by the vehicle (KN-62 + VEH) or melatonin (KN-62 + MEL) for 6 h. After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Slices were analyzed by the modified Sholl method. Results represent the mean ± SEM of one experiment of three done by quadruplicate. Asterisks show significant differences (<span class="html-italic">p</span> &lt; 0.05). (<b>A</b>) Number of primary dendrites; (<b>B</b>) Primary dendrite length (µm); (<b>C</b>) Number of secondary dendrites; (<b>D</b>) Secondary and tertiary dendrite length (µm).</p>
Full article ">Figure 4
<p>Relative content of phosphorylated Ca<sup>2+</sup>/CaM Kinase II, Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 in hippocampal slices incubated with Melatonin. Hippocampal slices were incubated for 3 h with either the vehicle (VEH) or 100 nM melatonin (MEL). CaMKII and Thr286-phospho-CaMKII (P-CaMKII), PKC and phospho-PKC (P-PKC) as well as ERK1/2 and phospho-ERK1/2 (P-ERK1/2) were determined by Western blot. Upper panels show immunodetection of GAPDH as load control. Representative fluorograms of total and phosphorylated enzymes revealed by ECL are shown below. Histograms correspond to densitometric analysis of bands depicted immediately above. Results are expressed as the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisks indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 5
<p>Calmodulin content in hippocampal slices treated with Melatonin. Hippocampal slices were incubated during 6 h with either the vehicle (VEH) or melatonin (MEL). Calmodulin (CaM) in the homogenates (<b>A</b>); and in the soluble (SOL) and cytoskeletal fractions (CSK) separated by centrifugation (<b>B</b>); was determined by Western blot. Upper panels show Carbonic Anhydrase (CA) used as external load control and stained with Coomassie blue. Representative fluorograms of CaM are shown immediately below. First lane of both gel and fluorogram from panel A was loaded with pure CA (5 µg) and CaM (1 µg), respectively. CaM was recognized with a specific CaM antibody and ECL. Histograms correspond to densitometric analysis of the bands shown in the upper panels. Results are the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisk indicates significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 6
<p>Participation of Melatonin receptors on dendrite formation elicited by Melatonin. To evaluate the involvement of MEL receptors, hippocampal organotypic cultures were incubated with the MT1/2 receptor antagonist, luzindole. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal<sup>®</sup> media for 7 days. Then they were incubated for 6 h with either the vehicle (<b>A</b>); 100 nM melatonin (<b>B</b>); or pre-incubated with 100 µM luzindole followed by 6 h incubation with the vehicle (<b>C</b>) or 100 nM melatonin (<b>D</b>). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Images were acquired with a camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.</p>
Full article ">Figure 6 Cont.
<p>Participation of Melatonin receptors on dendrite formation elicited by Melatonin. To evaluate the involvement of MEL receptors, hippocampal organotypic cultures were incubated with the MT1/2 receptor antagonist, luzindole. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal<sup>®</sup> media for 7 days. Then they were incubated for 6 h with either the vehicle (<b>A</b>); 100 nM melatonin (<b>B</b>); or pre-incubated with 100 µM luzindole followed by 6 h incubation with the vehicle (<b>C</b>) or 100 nM melatonin (<b>D</b>). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Images were acquired with a camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.</p>
Full article ">Figure 7
<p>Morphometric analysis of dendrite formation elicited with Melatonin in presence of Luzindole. Hippocampal slices were incubated for 6 h with either the vehicle (VEH), 100 nM melatonin (MEL), or were pre-incubated with luzindole (LZD) for 15 min followed by 6 h incubation with the vehicle (LZD + VEH) or 100 nM melatonin (LZD + MEL). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Slices were analyzed by the modified Sholl method. Results represent the mean ± SEM of one experiment of three done by quadruplicate. Asterisks show significant differences (<span class="html-italic">p</span> &lt; 0.05). (<b>A</b>) Number of primary dendrites; (<b>B</b>) Primary dendrite length (µm); (<b>C</b>) Number of secondary dendrites; (<b>D</b>) Secondary and tertiary dendrite length (µm).</p>
Full article ">Figure 7 Cont.
<p>Morphometric analysis of dendrite formation elicited with Melatonin in presence of Luzindole. Hippocampal slices were incubated for 6 h with either the vehicle (VEH), 100 nM melatonin (MEL), or were pre-incubated with luzindole (LZD) for 15 min followed by 6 h incubation with the vehicle (LZD + VEH) or 100 nM melatonin (LZD + MEL). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Slices were analyzed by the modified Sholl method. Results represent the mean ± SEM of one experiment of three done by quadruplicate. Asterisks show significant differences (<span class="html-italic">p</span> &lt; 0.05). (<b>A</b>) Number of primary dendrites; (<b>B</b>) Primary dendrite length (µm); (<b>C</b>) Number of secondary dendrites; (<b>D</b>) Secondary and tertiary dendrite length (µm).</p>
Full article ">Figure 8
<p>Schematic drawing of the modified Sholl method to determine the number and length of primary and secondary dendrites. A grid of concentric circles equidistantly drawn five micrometers apart was superimposed at the center of the neuronal soma (blue arrow). Dendrite formation was determined by counting the number of emerging dendrites from the soma. Primary dendrite length was measured from the soma up to the first intersection (green line). Complexity of dendrites was determined by counting the number of secondary dendrites as well as by measuring their length (red line).</p>
Full article ">Figure 9
<p>Schematic drawing of the signaling pathway by which Melatonin stimulates dendrite formation. Melatonin (MEL) binds to membrane receptors (MT2/MT1) to activate PKC through the diacylglycerol (DAG) pathway. Also, the indoleamine can diffuse through the plasmatic membrane to directly interact with protein kinase C (PKC). This enzyme may phosphorylate calmodulin (CaM), which can bind to Ca<sup>2<b>+</b></sup>/CaM Kinase II (CaMKII) with higher affinity. CaM-activated CaMKII undergoes autophosphorylation which induces its targeting to the cytoskeletal compartment where it binds and phosphorylates MAP2 to constitute dendrites. Also, PKC may increase CaM levels in the soluble fraction by an enhanced biosynthesis or by targeting from the cytoskeletal to the soluble compartment.</p>
Full article ">
2232 KiB  
Article
Isolation and Expression of NAC Genes during Persimmon Fruit Postharvest Astringency Removal
by Ting Min, Miao-Miao Wang, Hongxun Wang, Xiaofen Liu, Fang Fang, Donald Grierson, Xue-Ren Yin and Kun-Song Chen
Int. J. Mol. Sci. 2015, 16(1), 1894-1906; https://doi.org/10.3390/ijms16011894 - 15 Jan 2015
Cited by 25 | Viewed by 7204
Abstract
NAC genes have been characterized in numerous plants, where they are involved in responses to biotic and abiotic stress, including low oxygen stress. High concentration of CO2 is one of the most effective treatments to remove astringency of persimmon fruit owing to [...] Read more.
NAC genes have been characterized in numerous plants, where they are involved in responses to biotic and abiotic stress, including low oxygen stress. High concentration of CO2 is one of the most effective treatments to remove astringency of persimmon fruit owing to the action of the accumulated anoxia metabolite acetaldehyde. In model plants, NAC genes have been identified as being responsive to low oxygen. However, the possible relationship between NAC transcription factors and persimmon astringency removal remains unexplored. In the present research, treatment with a high concentration of CO2 (95%) effectively removed astringency of “Mopan” persimmon fruit by causing decreases in soluble tannin. Acetaldehyde content increased in response to CO2 treatment concomitantly with astringency removal. Using RNA-seq and Rapid amplification of cDNA ends (RACE), six DkNAC genes were isolated and studied. Transcriptional analysis indicated DkNAC genes responded differentially to CO2 treatment; DkNAC1, DkNAC3, DkNAC5 and DkNAC6 were transiently up-regulated, DkNAC2 was abundantly expressed 3 days after treatment, while the DkNAC4 was suppressed during astringency removal. It is proposed that DkNAC1/3/5/6 could be important candidates as regulators of persimmon astringency removal and the roles of other member are also discussed. Full article
Show Figures

Figure 1

Figure 1
<p>Effect of CO<sub>2</sub> treatment on soluble tannin of “Mopan” fruit at 20 °C. Mature fruit were treated with CO<sub>2</sub> (~95%, <span class="html-italic">v</span>/<span class="html-italic">v</span>, open circles, one day) and air (control, open squares), separately. Error bars represent standard error from three biological replicates.</p>
Full article ">Figure 2
<p>Comparison of tannin printing of control and CO<sub>2</sub> treated “Mopan” fruit at 2 days in storage.</p>
Full article ">Figure 3
<p>Effect of CO<sub>2</sub> treatment on acetaldehyde content of “Mopan” fruit. Mature fruit were treated with CO<sub>2</sub> (~95%, <span class="html-italic">v</span>/<span class="html-italic">v</span>, white bars, one day) and air (control, black bars), separately at 20 °C. Error bars represent standard error from three biological replicates.</p>
Full article ">Figure 4
<p>Amino acid sequence alignment of the DkNAC proteins with <span class="html-italic">Arabidopsis</span> and rice NAC proteins. DkNAC proteins were aligned with <span class="html-italic">Arabidopsis</span> ANAC019 (At1g52890.1), ANAC042 (At2g43000.1), ANAC043 (At2g46770.1), ANAC102 (AT5G63790.1) and rice ONAC022 (AK107090). Identical and similar amino acids are indicated by black and grey shading, respectively. Gaps were introduced to optimize alignment. The five highly conserved amino acid motifs (<b>A</b>–<b>E</b>) are indicated by black lines.</p>
Full article ">Figure 5
<p>Phylogenetic tree of <span class="html-italic">NAC</span> genes. Persimmon <span class="html-italic">DkNAC</span> genes are highlighted in red. The amino acid sequences of the <span class="html-italic">Arabidopsis ERF</span> family were obtained from TAIR. The phylogenetic tree was constructed with figtree (version 3.1).</p>
Full article ">Figure 6
<p>Transcriptional analysis of <span class="html-italic">DkNAC</span> genes. Transcripts of <span class="html-italic">DkNAC</span> genes were measured by real-time PCR. Fruit were treated with 95% CO<sub>2</sub> for one day at 20 °C in sealed container, while control fruit was sealed in a similar container without any treatment. Day 0 fruit values were set as 1. Error bars indicate standard error from three biological replicates.</p>
Full article ">
1327 KiB  
Review
Surveillance and Cleavage of Eukaryotic tRNAs
by Cyrille Megel, Geoffrey Morelle, Stéphanie Lalande, Anne-Marie Duchêne, Ian Small and Laurence Maréchal-Drouard
Int. J. Mol. Sci. 2015, 16(1), 1873-1893; https://doi.org/10.3390/ijms16011873 - 15 Jan 2015
Cited by 58 | Viewed by 10091
Abstract
Beyond their central role in protein synthesis, transfer RNAs (tRNAs) have many other crucial functions. This includes various roles in the regulation of gene expression, stress responses, metabolic processes and priming reverse transcription. In the RNA world, tRNAs are, with ribosomal RNAs, among [...] Read more.
Beyond their central role in protein synthesis, transfer RNAs (tRNAs) have many other crucial functions. This includes various roles in the regulation of gene expression, stress responses, metabolic processes and priming reverse transcription. In the RNA world, tRNAs are, with ribosomal RNAs, among the most stable molecules. Nevertheless, they are not eternal. As key elements of cell function, tRNAs need to be continuously quality-controlled. Two tRNA surveillance pathways have been identified. They act on hypo-modified or mis-processed pre-tRNAs and on mature tRNAs lacking modifications. A short overview of these two pathways will be presented here. Furthermore, while the exoribonucleases acting in these pathways ultimately lead to complete tRNA degradation, numerous tRNA-derived fragments (tRFs) are present within a cell. These cleavage products of tRNAs now potentially emerge as a new class of small non-coding RNAs (sncRNAs) and are suspected to have important regulatory functions. The tRFs are evolutionarily widespread and created by cleavage at different positions by various endonucleases. Here, we review our present knowledge on the biogenesis and function of tRFs in various organisms. Full article
(This article belongs to the Special Issue Functions of Transfer RNAs)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Overview of the two tRNA degradation pathways of <span class="html-italic">S. cerevisae.</span> This figure is adapted from [<a href="#B10-ijms-16-01873" class="html-bibr">10</a>]. (<b>A</b>) The tRNA nuclear surveillance pathway was primarily shown to act on pre-tRNAs lacking modifications (e.g., pre-tRNA<sub>i</sub><sup>Met</sup>). Hypo-modified pre-tRNA is first polyadenylated by the TRAMP complex and then degraded by the exosome; (<b>B</b>) Degradation of mature tRNAs through the rapid tRNA decay (RTD) pathway. RTD was shown to act on hypo-modified tRNAs (marked with red crosses) (e.g., tRNA<sup>Val(AAC)</sup>). The major components are depicted. The implication of the tRNA-nucleotidyl transferase (CCAse) enzyme in the RTD pathway is also presented [<a href="#B33-ijms-16-01873" class="html-bibr">33</a>]. A, CCA triplet or a short poly(A) tail (AAAA) depicted in red can be added by the CCAse enzyme.</p>
Full article ">Figure 2
<p>Summary of our knowledge on the major identified molecular functions assigned to tRFs. References for the functional implications of tRFs are the following: (<b>A</b>) [<a href="#B89-ijms-16-01873" class="html-bibr">89</a>]; (<b>B</b>) [<a href="#B56-ijms-16-01873" class="html-bibr">56</a>]; (<b>C</b>) [<a href="#B97-ijms-16-01873" class="html-bibr">97</a>]; (<b>D</b>) [<a href="#B73-ijms-16-01873" class="html-bibr">73</a>]; (<b>E</b>) [<a href="#B74-ijms-16-01873" class="html-bibr">74</a>]; (<b>F</b>) [<a href="#B98-ijms-16-01873" class="html-bibr">98</a>]; (<b>G</b>) [<a href="#B99-ijms-16-01873" class="html-bibr">99</a>]; (<b>H</b>) [<a href="#B100-ijms-16-01873" class="html-bibr">100</a>]. Straight arrows indicate the generated tRFs and bent arrows their functions. Dashed arrow indicates potential functions.</p>
Full article ">
2696 KiB  
Article
Structural Diversity of the Microbial Surfactin Derivatives from Selective Esterification Approach
by Chuanshi Shao, Lin Liu, Hongze Gang, Shizhong Yang and Bozhong Mu
Int. J. Mol. Sci. 2015, 16(1), 1855-1872; https://doi.org/10.3390/ijms16011855 - 15 Jan 2015
Cited by 29 | Viewed by 7566
Abstract
Surfactin originated from genus Bacillus is composed of a heptapeptide moiety bonded to the carboxyl and hydroxyl groups of a ?-hydroxy fatty acid and it can be chemically modified to prepare the derivatives with different structures, owing to the existence of two free [...] Read more.
Surfactin originated from genus Bacillus is composed of a heptapeptide moiety bonded to the carboxyl and hydroxyl groups of a ?-hydroxy fatty acid and it can be chemically modified to prepare the derivatives with different structures, owing to the existence of two free carboxyl groups in its peptide loop. This article presents the chemical modification of surfactin esterified with three different alcohols, and nine novel surfactin derivatives have been separated from products by the high performance liquid chromatography (HPLC). The novel derivatives, identified with Fourier transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectrometry (ESI-MS), are the mono-hexyl-surfactin C14 ester, mono-hexyl-surfactin C15 ester, mono-2-methoxy-ethyl-surfactin C14 ester, di-hexyl-surfactin C14 ester, di-hexyl-surfactin ester C15, di-2-methoxy-ethyl-surfactin ester C14, di-2-methoxy-ethyl-surfactin ester C15, di-6-hydoxyl-hexyl-surfactin C14 ester and, di-6-hydoxyl-hexyl-surfactin C15 ester. The reaction conditions for esterification were optimized and the dependence of yields on different alcohols and catalysts were discussed. This study shows that esterification is one of the most efficient ways of chemical modification for surfactin and it can be used to prepare more derivatives to meet the needs of study in biological and interfacial activities. Full article
(This article belongs to the Section Green Chemistry)
Show Figures

Figure 1

Figure 1
<p>The structure of surfactin C13 (R = C<sub>4</sub>H<sub>9</sub>), C14 (R = C<sub>5</sub>H<sub>11</sub>), C15 (R = C<sub>6</sub>H<sub>13</sub>) [<a href="#B7-ijms-16-01855" class="html-bibr">7</a>].</p>
Full article ">Figure 2
<p>High performance liquid chromatography (HPLC) spectra of surfactin (dashed line) and surfactin-(Glu-γ, Asp-β)-hexyl ester (solid line) under the same condition.</p>
Full article ">Figure 3
<p>Fourier transform infrared spectroscopy (FT-IR) results of surfactin-C14, surfactin-C15, B1, B2, B3 and B4. (<b>A</b>) for surfactin-C14 and mono-hexyl-surfactin C14 ester; (<b>B</b>) for surfactin-C15 and mono-hexyl-surfactin C15 ester; (<b>C</b>) for surfactin-C14 and <span class="html-italic">di</span>-hexyl-surfactin C14 ester; and (<b>D</b>) for surfactin-C15 and <span class="html-italic">di</span>-hexyl-surfactin C15 ester.</p>
Full article ">Figure 3 Cont.
<p>Fourier transform infrared spectroscopy (FT-IR) results of surfactin-C14, surfactin-C15, B1, B2, B3 and B4. (<b>A</b>) for surfactin-C14 and mono-hexyl-surfactin C14 ester; (<b>B</b>) for surfactin-C15 and mono-hexyl-surfactin C15 ester; (<b>C</b>) for surfactin-C14 and <span class="html-italic">di</span>-hexyl-surfactin C14 ester; and (<b>D</b>) for surfactin-C15 and <span class="html-italic">di</span>-hexyl-surfactin C15 ester.</p>
Full article ">Figure 4
<p>Electrospray ionization mass spectrometry (ESI-MS) spectroscopy of B1–B4.</p>
Full article ">Figure 5
<p>The structures of B1, B2, B3 and B4. B1: R<sub>1</sub> = C<sub>5</sub>H<sub>11</sub>, R<sub>2</sub> = C<sub>4</sub>H<sub>9</sub>, R<sub>3</sub> = H or R<sub>1</sub> = C<sub>5</sub>H<sub>11</sub>, R<sub>2</sub> = H, R<sub>3</sub> = C<sub>6</sub>H<sub>13</sub>; B2: R<sub>1</sub> = C<sub>6</sub>H<sub>13</sub>, R<sub>2</sub> = C<sub>6</sub>H<sub>13</sub>, R<sub>3</sub> = H or R<sub>1</sub> = C<sub>6</sub>H<sub>13</sub>, R<sub>2</sub> = H, R<sub>3</sub> = C<sub>6</sub>H<sub>13</sub>; and B3: R<sub>1</sub> = C<sub>5</sub>H<sub>11</sub>, R<sub>2</sub> = C<sub>6</sub>H<sub>13</sub>, R<sub>3</sub> = C<sub>6</sub>H<sub>13</sub>; B4: R<sub>1</sub> = C<sub>6</sub>H<sub>13</sub>, R<sub>2</sub> = C<sub>6</sub>H<sub>13</sub>, R<sub>3</sub> = C<sub>6</sub>H<sub>13</sub>.</p>
Full article ">Figure 6
<p>HPLC spectra of surfactin (dashed line) and (Glu-γ, Asp-β)-2-methoxy-ethyl-surfactin ester (solid line) under the same condition.</p>
Full article ">Figure 7
<p>FT-IR spectra of E1, E2 and E3. (<b>A</b>) for the surfactin-C14 and mono-2-methoxy-ethyl-surfactin-C14 ester (E1); (<b>B</b>) for the surfactin-C14 and <span class="html-italic">di</span>-2-methoxy-ethyl-surfactin C14 ester; and (<b>C</b>) for surfactin-C15 and <span class="html-italic">di</span>-2-methoxy-ethyl-surfactin C14 ester.</p>
Full article ">Figure 8
<p>ESI-MS spectroscopy of E1, E2 and E3.</p>
Full article ">Figure 9
<p>The structures of E1, E2 and E3. E1: R<sub>1</sub> = C<sub>5</sub>H<sub>11</sub>, R<sub>2</sub> = CH<sub>2</sub>–CH<sub>2</sub>–O–CH<sub>3</sub>, R<sub>3</sub> = H or R<sub>1</sub> = C<sub>5</sub>H<sub>11</sub>, R2 =H, R<sub>3</sub> = CH<sub>2</sub>–CH<sub>2</sub>–O–CH<sub>3</sub>; E2: R<sub>1</sub> = C<sub>5</sub>H<sub>11</sub>, R<sub>2</sub> = CH<sub>2</sub>–CH<sub>2</sub>–O–CH<sub>3</sub>, R<sub>3</sub> = CH<sub>2</sub>–CH<sub>2</sub>–O–CH<sub>3</sub>; and E3: R<sub>1</sub> = C<sub>6</sub>H<sub>13</sub>, R<sub>2</sub> = CH<sub>2</sub>–CH<sub>2</sub>–O–CH<sub>3</sub>, R<sub>3</sub> = CH<sub>2</sub>–CH<sub>2</sub>–O–CH<sub>3</sub>.</p>
Full article ">Figure 10
<p>HPLC spectra of surfactin (dashed line) and (Glu-γ, Asp-β)-6-hydoxyl-hexyl-surfactin ester (solid line).</p>
Full article ">Figure 11
<p>FT-IR results of surfactin-C14 and surfactin-C15, D1 and D2. (<b>A</b>) for surfactin-C14 and <span class="html-italic">di</span>-6-hydoxyl-hexyl-surfactin C14 ester; and (<b>B</b>) for surfactin-C15 and <span class="html-italic">di</span>-6-hydoxyl-hexyl-surfactin C15 ester.</p>
Full article ">Figure 12
<p>ESI-MS spectroscopy of D1 and D2.</p>
Full article ">Figure 13
<p>The structures of D1 and D2. D1: R = C<sub>5</sub>H<sub>11</sub>; D2: R = C<sub>6</sub>H<sub>13</sub>.</p>
Full article ">Figure 14
<p>The ratio of (<b>A</b>) monoester-SF and (<b>B</b>) diester-SF in the product in four reaction conditions. Condition 1: catalyzed by 1 mol/L HCl; reacted for 24 h; Condition 2: catalyzed by 1 mol/L HCl; reacted for 48 h; Condition 3: catalyzed by concentrated HCl; reacted for 24 h; Condition 4: catalyzed by concentrated HCl; reacted for 48 h.</p>
Full article ">
837 KiB  
Article
Capability of Utilizing CYP3A5 Polymorphisms to Predict Therapeutic Dosage of Tacrolimus at Early Stage Post-Renal Transplantation
by Takenori Niioka, Hideaki Kagaya, Mitsuru Saito, Takamitsu Inoue, Kazuyuki Numakura, Tomonori Habuchi, Shigeru Satoh and Masatomo Miura
Int. J. Mol. Sci. 2015, 16(1), 1840-1854; https://doi.org/10.3390/ijms16011840 - 14 Jan 2015
Cited by 14 | Viewed by 6805
Abstract
While CYP3A5 polymorphisms are used to predict the initial dosage of tacrolimus therapy, the predictive capability of genetic information for dosing at early stage post-renal transplantation is unknown. We investigated the influence of polymorphisms over time. An initial oral dose of modified-release once-daily [...] Read more.
While CYP3A5 polymorphisms are used to predict the initial dosage of tacrolimus therapy, the predictive capability of genetic information for dosing at early stage post-renal transplantation is unknown. We investigated the influence of polymorphisms over time. An initial oral dose of modified-release once-daily tacrolimus formulation (0.20 mg/kg) was administered to 50 Japanese renal transplant patients every 24 h. Stepwise multiple linear regression analysis for tacrolimus dosing was performed each week to determine the effect of patient clinical characteristics. The dose-adjusted trough concentration was approximately 70% higher for patients with the CYP3A5*3/*3 than patients with the CYP3A5*1 allele before the second pre-transplantation tacrolimus dose (0.97 (0.78–1.17) vs. 0.59 (0.45–0.87) ng/mL/mg; p < 0.001). The contribution of genetic factors (CYP3A5*1 or *3) for tacrolimus dosing showed increased variation from Day 14 to Day 28 after transplantation: 7.2%, 18.4% and 19.5% on Days 14, 21 and 28, respectively. The influence of CYP3A5 polymorphisms on the tacrolimus maintenance dosage became evident after Day 14 post-transplantation, although the tacrolimus dosage was determined based only on patient body weight for the first three days after surgery. Tacrolimus dosage starting with the initial administration should be individualized using the CYP3A5 genotype information. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
Show Figures

Figure 1

Figure 1
<p>Comparison of dose-adjusted trough concentrations (<span class="html-italic">C</span><sub>24h</sub>/dose) just prior to the second dose of tacrolimus pre-transplantation between <span class="html-italic">CYP3A5</span> extensive metabolizer (EM) (white boxes) and poor metabolizer (PM) (gray boxes) patients. Graphical analysis was performed using an SPSS box and whiskers plot. The box spans data between two quartiles (IQR), with the median represented as a bold horizontal line. The ends of the whiskers (vertical lines) represent the smallest and largest values that were not outliers. Outliers (circles) are values between 1.5 and 3 IQRs from the end of the box. Values of more than three IQRs from the end of the box are defined as extreme (asterisk).</p>
Full article ">Figure 2
<p>Percentage of dose variation for once-daily formulation of tacrolimus determined at weekly time points. CL<sub>t</sub> during CIV; total clearance of tacrolimus during continuous intravenous infusion (CIV). PO; oral administration. EM, <span class="html-italic">CYP3A5</span><span class="html-italic">*1</span>/<span class="html-italic">*1</span> + <span class="html-italic">*1</span>/<span class="html-italic">*3</span>; PM, <span class="html-italic">CYP3A5</span><span class="html-italic">*3</span>/<span class="html-italic">*3</span>.</p>
Full article ">Figure 3
<p>Comparison of achievement rates from the initial <span class="html-italic">C</span><sub>0h</sub> to the target range (target <span class="html-italic">C</span><sub>0h</sub> ± 20%) between <span class="html-italic">CYP3A5</span> genotypes at each stage after renal transplantation. White columns, lower than the target range; black columns, within the target range; gray columns; more than the target range. EM (<span class="html-italic">*1</span>/<span class="html-italic">*1</span> + <span class="html-italic">*1</span>/<span class="html-italic">*3</span>), <span class="html-italic">n</span> = 17; PM (<span class="html-italic">*3</span>/<span class="html-italic">*3</span>), <span class="html-italic">n</span> = 33.</p>
Full article ">Figure 4
<p>Comparison of body weight-adjusted dosage and trough concentration (open circles) of tacrolimus on Day 28 after surgery between <span class="html-italic">CYP3A5</span> EM and PM patients. Graphical analysis was performed using an SPSS box and whiskers plot. The box spans data between two quartiles (IQR), with the median represented as a bold horizontal line. The ends of the whiskers (vertical lines) represent the smallest and largest values that were not outliers.</p>
Full article ">
1721 KiB  
Article
Non-Classical Gluconeogenesis-Dependent Glucose Metabolism in Rhipicephalus microplus Embryonic Cell Line BME26
by Renato Martins Da Silva, Bárbara Della Noce, Camila Fernanda Waltero, Evenilton Pessoa Costa, Leonardo Araujo De Abreu, Naftaly Wang'ombe Githaka, Jorge Moraes, Helga Fernandes Gomes, Satoru Konnai, Itabajara Da Silva Vaz, Jr., Kazuhiko Ohashi and Carlos Logullo
Int. J. Mol. Sci. 2015, 16(1), 1821-1839; https://doi.org/10.3390/ijms16011821 - 14 Jan 2015
Cited by 19 | Viewed by 7020
Abstract
In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways [...] Read more.
In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells. Full article
(This article belongs to the Section Biochemistry)
Show Figures

Figure 1

Figure 1
<p>Transcriptional and activities of glycolytic enzymes are glucose concentration-dependent in BME26 cells. Transcriptional analysis of <span class="html-italic">hexokinase</span> (<b>A</b>); <span class="html-italic">pyruvate kinase</span> (<b>B</b>); HK activity (<b>C</b>) and pyruvate kinase activity (<b>D</b>), glycolytic key-enzymes, in embryonic <span class="html-italic">Rhipicephalus microplus</span> cells (BME26) in response to glucose treatment. Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (<b>*</b> <span class="html-italic">p</span> &lt; 0.05; <b>**</b> <span class="html-italic">p</span> &lt; 0.001, ANOVA).</p>
Full article ">Figure 2
<p>Higher oxygen consumption under high-glucose treatment. The oxygen consumption rate was measured in embryonic <span class="html-italic">Rhipicephalus microplus</span> cells (BME26) in response to glucose treatment. Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (<b>*</b> <span class="html-italic">p</span> &lt; 0.05; <b>**</b> <span class="html-italic">p</span> &lt; 0.001, ANOVA).</p>
Full article ">Figure 3
<p>Glucose availability has an essential role in BME26 cell survival. Cell viability was performed in embryonic <span class="html-italic">Rhipicephalus microplus</span> cells (BME26) in response to glucose treatment. Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (<b>**</b> <span class="html-italic">p</span> &lt; 0.001, ANOVA).</p>
Full article ">Figure 4
<p>Membrane integrity is unaffected in BME26 cells after glucose treatment. The cells were directly stained by adding Hoechst 33342 and propidium iodide. Glass slides were observed in a fluorescence microscope (model Eclipse 80i, Nikon), and pictures were obtained at 400× magnification. Control: cells maintained with 50 mM of glucose (<b>A</b>); Low: cell maintained without glucose addition (<b>B</b>); and High: cells maintained with 100 mM of glucose (<b>C</b>). The arrows indicate the shape of low-glucose cells and the triangles indicate the rounded shape of high-glucose cells. Scale bar: 10 μm.</p>
Full article ">Figure 5
<p>Glycogen metabolism synthesis has a transcriptional control in BME26 cells. Transcriptional analysis of <span class="html-italic">glycogen synthase</span> (<b>A</b>) and <span class="html-italic">glycogen synthase kinase 3</span> (<b>B</b>) in embryonic <span class="html-italic">Rhipicephalus microplus</span> cells (BME26) in response to glucose treatment. Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (<b>*</b> <span class="html-italic">p</span> &lt; 0.05; <b>**</b> <span class="html-italic">p</span> &lt; 0.001, ANOVA).</p>
Full article ">Figure 6
<p>Glycogen degradation has a transcriptional control in BME26 cells. Transcriptional analysis of <span class="html-italic">glycogen debranching enzyme</span> (<b>A</b>) and <span class="html-italic">phosphoglucomutase</span> (<b>B</b>) in embryonic <span class="html-italic">Rhipicephalus microplus</span> cells (BME26) in response to glucose treatment. Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (<b>*</b> <span class="html-italic">p</span> &lt; 0.05; <b>**</b> <span class="html-italic">p</span> &lt; 0.001, ANOVA).</p>
Full article ">Figure 7
<p>Gluconeogenic response increase in high glucose concentration in BME26 cells. Transcriptional analysis of PEPCK (<b>A</b>) and <span class="html-italic">glucose-6-phosphatase</span> (<b>B</b>), gluconeogenic key-enzymes, in embryonic <span class="html-italic">Rhipicephalus microplus</span> cells (BME26) in response to glucose treatment. Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (<b>**</b> <span class="html-italic">p</span> &lt; 0.001, ANOVA).</p>
Full article ">Figure 8
<p>Gluconeogenic enzymes have transcriptional control by GSK3 in BME26 silenced-cells. Transcriptional analysis of PEPCK (<b>A</b>) and glucose-6 phosphatase (<b>B</b>), gluconeogenic key-enzymes, in embryonic <span class="html-italic">Rhipicephalus microplus</span> cells (BME26) in response to GSK3 silencing (<b>C</b>). Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (<b>**</b> <span class="html-italic">p</span> &lt; 0.001, paired T test).</p>
Full article ">Figure 9
<p>Scheme for pathways of glucose metabolism. The scheme is based on enzyme activities, metabolites and molecular changes in BME26 cells after glucose treatments. The enzymes studied are presented inside blue spheres (HK, PK, GS, GSK3, PGM, GDE, PEPCK and GP). Arrows next to the enzymes (↓↑) represent the oscillations in enzymatic activity and transcriptional response. Small spheres next enzymes (○) indicate no variations in these enzymes or genes compared with the control. Dashed line involves several enzymes steps in the pathway, and solid lid represents one enzymatic step. The low-glucose (<b>B</b>) and high-glucose (<b>C</b>) are compared with the control (<b>A</b>).</p>
Full article ">
1626 KiB  
Article
Specificity Protein 1 Regulates Gene Expression Related to Fatty Acid Metabolism in Goat Mammary Epithelial Cells
by Jiangjiang Zhu, Yuting Sun, Jun Luo, Min Wu, Jianhua Li and Yanhong Cao
Int. J. Mol. Sci. 2015, 16(1), 1806-1820; https://doi.org/10.3390/ijms16011806 - 14 Jan 2015
Cited by 60 | Viewed by 7118
Abstract
Specificity protein 1 (SP1) is a ubiquitous transcription factor that plays an important role in controlling gene expression. Although important in mediating the function of various hormones, the role of SP1 in regulating milk fat formation remains unknown. To investigate the sequence and [...] Read more.
Specificity protein 1 (SP1) is a ubiquitous transcription factor that plays an important role in controlling gene expression. Although important in mediating the function of various hormones, the role of SP1 in regulating milk fat formation remains unknown. To investigate the sequence and expression information, as well as its role in modulating lipid metabolism, we cloned SP1 gene from mammary gland of Xinong Saanen dairy goat. The full-length cDNA of the SP1 gene is 4376 bp including 103 bp of 5'UTR, 2358 bp of ORF (HM_236311) and 1915 bp of 3'UTR, which is predicted to encode a 786 amino acids polypeptide. Phylogenetic tree analysis showed that goat SP1 has the closest relationship with sheep, followed by bovines (bos taurus, odobenus and ceratotherium), pig, primates (pongo, gorilla, macaca and papio) and murine (rattus and mus), while the furthest relationship was with canis and otolemur. Expression was predominant in the lungs, small intestine, muscle, spleen, mammary gland and subcutaneous fat. There were no significant expression level differences between the mammary gland tissues collected at lactation and dry-off period. Overexpression of SP1 in goat mammary epithelial cells (GMECs) led to higher mRNA expression level of peroxisome proliferator-activated receptor-? (PPAR?) and lower liver X receptor ? (LXR?) mRNA level, both of which were crucial in regulating fatty acid metabolism, and correspondingly altered the expression of their downstream genes in GMECs. These results were further enhanced by the silencing of SP1. These findings suggest that SP1 may play an important role in fatty acid metabolism. Full article
(This article belongs to the Section Biochemistry)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Structure prediction of SP1 proteins. (<b>A</b>) The secondary structure of SP1 protein. Blue color represents alpha helixes, red color represents beta bridges, and purple represents random coils; (<b>B</b>) The tertiary structure of SP1 protein in various species.</p>
Full article ">Figure 2
<p>Phylogenetic tree based on <span class="html-italic">SP1</span> gene sequences of 31 representative animals made with MEGA 5 software (S. Kumar, Tempe, AZ, USA) using Neighbor-Joining (NJ) method.</p>
Full article ">Figure 3
<p>The mRNA expression profiles of <span class="html-italic">SP1</span>. (<b>A</b>) mRNA expression of <span class="html-italic">SP1</span> in various tissues in dairy goats; (<b>B</b>) Expression level of <span class="html-italic">SP1</span> between lactation and dry-off periods.</p>
Full article ">Figure 4
<p>Effect of expression alteration of SP1 on the expression of genes relative to lipid metabolism. (<b>A</b>) Ad-<span class="html-italic">SP1</span> overexpressed the expression level of SP1 protein; (<b>B</b>) Ad-<span class="html-italic">SP1</span> overexpressed the mRNA expression level of the <span class="html-italic">SP1</span> gene; (<b>C</b>) Overexpression of <span class="html-italic">SP1</span> altered the mRNA expression level of genes related to lipid metabolism in goat mammary epithelial cells (GMECs); (<b>D</b>) siRNA decreased the protein expression of SP1; (<b>E</b>) siRNA decreased the mRNA expression of <span class="html-italic">SP1</span>; and (<b>F</b>) The silencing of <span class="html-italic">SP1</span> affected the mRNA expression of genes associated with lipid metabolism. Columns, average of 3 repeats; bars, SD; *, <span class="html-italic">p</span> &lt; 0.05</p>
Full article ">
1358 KiB  
Review
Cellular Disulfide Bond Formation in Bioactive Peptides and Proteins
by Nitin A. Patil, Julien Tailhades, Richard Anthony Hughes, Frances Separovic, John D. Wade and Mohammed Akhter Hossain
Int. J. Mol. Sci. 2015, 16(1), 1791-1805; https://doi.org/10.3390/ijms16011791 - 14 Jan 2015
Cited by 55 | Viewed by 22637
Abstract
Bioactive peptides play important roles in metabolic regulation and modulation and many are used as therapeutics. These peptides often possess disulfide bonds, which are important for their structure, function and stability. A systematic network of enzymes—a disulfide bond generating enzyme, a disulfide bond [...] Read more.
Bioactive peptides play important roles in metabolic regulation and modulation and many are used as therapeutics. These peptides often possess disulfide bonds, which are important for their structure, function and stability. A systematic network of enzymes—a disulfide bond generating enzyme, a disulfide bond donor enzyme and a redox cofactor—that function inside the cell dictates the formation and maintenance of disulfide bonds. The main pathways that catalyze disulfide bond formation in peptides and proteins in prokaryotes and eukaryotes are remarkably similar and share several mechanistic features. This review summarizes the formation of disulfide bonds in peptides and proteins by cellular and recombinant machinery. Full article
(This article belongs to the Special Issue Bioactive Carbohydrates and Peptides)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Schematic representation of general mechanisms by which a disulfide bond is formed by an intra-cellular enzyme-cofactor system: (<b>A</b>) Formation of new disulfide bonds; and (<b>B</b>) reshuffling of existing bonds by isomerase activity.</p>
Full article ">Figure 2
<p>Cellular representation of enzyme systems and respective organelles.</p>
Full article ">
1916 KiB  
Review
Mechanisms and Implications of Dual-Acting Methotrexate in Folate-Targeted Nanotherapeutic Delivery
by Pamela T. Wong and Seok Ki Choi
Int. J. Mol. Sci. 2015, 16(1), 1772-1790; https://doi.org/10.3390/ijms16011772 - 13 Jan 2015
Cited by 75 | Viewed by 12666
Abstract
The rational design of a nanoplatform in drug delivery plays a crucial role in determining its targeting specificity and efficacy in vivo. A conventional approach relies on the surface conjugation of a nanometer-sized particle with two functionally distinct types of molecules, one [...] Read more.
The rational design of a nanoplatform in drug delivery plays a crucial role in determining its targeting specificity and efficacy in vivo. A conventional approach relies on the surface conjugation of a nanometer-sized particle with two functionally distinct types of molecules, one as a targeting ligand, and the other as a therapeutic agent to be delivered to the diseased cell. However, an alternative simplified approach can be used, in which a single type of molecule displaying dual function as both a targeting ligand and therapeutic agent is conjugated to the nanoparticle. In this review, we evaluate the validity of this new strategy by using methotrexate, which displays multifunctional mechanisms of action. Methotrexate binds to the folate receptor, a surface biomarker frequently overexpressed in tumor cells, and also inhibits dihydrofolate reductase, an enzyme critical for cell survival and division. Thus we describe a series of fifth generation poly(amido amine) dendrimers conjugated with methotrexate, and discuss several lines of evidence supporting the efficacy of this new platform strategy based on surface plasmon resonance spectroscopy, enzyme activity assays, and cell-based studies with folate receptor (+) KB cancer cells. Full article
(This article belongs to the Special Issue Bioactive Nanoparticles 2014)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Two strategies for targeted drug delivery to a folate receptor (FAR)-overexpressing tumor cell with a fifth generation (G5) dendrimer NP. (<b>A</b>) A conventional two-molecule approach with G5(FA)<sub>n</sub>(MTX)<sub>m</sub> presenting folic acid (FA) as a targeting ligand and carrying methotrexate (MTX) as a drug payload and (<b>B</b>) A dual-acting, single molecule approach with G5(MTX)<sub>n</sub> presenting MTX as both a targeting ligand and drug payload.</p>
Full article ">Figure 2
<p>(<b>A</b>) Structures of folic acid (FA) and methotrexate (MTX); (<b>B</b>,<b>C</b>) The crystal structure of human folate receptor beta (FOLR2) bound with FA or MTX at its ligand site (PDB code 4KNO) [<a href="#B67-ijms-16-01772" class="html-bibr">67</a>]. Inset: an expanded view of each bound ligand molecule. Surface images of the receptor-ligand complexes were generated by PyMOL™ (version 1.3).</p>
Full article ">Figure 3
<p>(<b>A</b>) Structure of G5(MTX)<sub>n</sub> (<span class="html-italic">n</span> = 5, 10) dendrimer conjugated with MTX via cyclooctyne-azide click chemistry; (<b>B</b>) Schematic for binding of FA, MTX and G5(MTX)<sub>n</sub> to the surface of a folate binding protein (FBP)-immobilized CM5 sensor chip; (<b>C</b>) Overlaid dose-dependent SPR sensorgrams [<a href="#B51-ijms-16-01772" class="html-bibr">51</a>].</p>
Full article ">Figure 4
<p>(<b>A</b>,<b>B</b>) Confocal microscopy images showing FAR-specific and time-dependent binding and uptake of (TAMRA)G5(MTX)<sub>10</sub> in FAR(+) KB and FAR(−) B16-F10 cells [<a href="#B51-ijms-16-01772" class="html-bibr">51</a>,<a href="#B52-ijms-16-01772" class="html-bibr">52</a>]; (<b>C</b>) Flow cytometric analysis. Inset: histograms showing the FL2 fluorescence (FL) of 10,000 KB or B16 cells. TAMRA = 5-carboxytetramethylrhodamine. Staining: Nuclei (DAPI; blue); Cytosol (TAMRA; red).</p>
Full article ">Figure 5
<p>(<b>A</b>,<b>B</b>) A crystal structure of a MTX molecule in complex with dihydrofolate reductase (DHFR) at its catalytic site (PDB code 1DDS [<a href="#B80-ijms-16-01772" class="html-bibr">80</a>], 1U72 [<a href="#B81-ijms-16-01772" class="html-bibr">81</a>]). The protein (surface, cartoon) and MTX molecule (stick) model of the enzyme-drug complex was generated by PyMOL™ (version 1.3); (<b>C</b>) Inhibition of human DHFR enzyme activity by MTX and G5(MTX)<sub>n</sub> (<span class="html-italic">n</span> = 0, 10; <a href="#ijms-16-01772-f003" class="html-fig">Figure 3</a>) conducted in a standard enzyme assay [<a href="#B52-ijms-16-01772" class="html-bibr">52</a>]. The concentrations on the X-axis are expressed as MTX or MTX equivalents rather than dendrimer.</p>
Full article ">Figure 6
<p><span class="html-italic">In vitro</span> cytotoxicity of G5(MTX)<sub>n</sub> in FAR(+) KB cells determined by an XTT assay [<a href="#B78-ijms-16-01772" class="html-bibr">78</a>]. Each data point represents a mean value (±SD). Doses for each conjugate on the <span class="html-italic">X</span>-axis are expressed as either dendrimer (<b>A</b>) or MTX (<b>B</b>) concentration.</p>
Full article ">Figure 7
<p>(<b>A</b>) Proposed schematic illustrating the concept of light-triggered MTX release; (<b>B</b>) <span class="html-italic">In vitro</span> cytotoxicity of G5(FA)<sub>9</sub>(MTX<b>*</b>)<sub>17</sub> in FAR(+) KB cells, before (control; <span class="html-italic">t</span> = 0) and after UV irradiation (<span class="html-italic">t</span> = 6 or 14 min) [<a href="#B84-ijms-16-01772" class="html-bibr">84</a>]. MTX<b>*</b> = MTX linked with a photocleavable linker.</p>
Full article ">
6491 KiB  
Article
Novel Transcription Factor Variants through RNA-Sequencing: The Importance of Being “Alternative”
by Margherita Scarpato, Antonio Federico, Alfredo Ciccodicola and Valerio Costa
Int. J. Mol. Sci. 2015, 16(1), 1755-1771; https://doi.org/10.3390/ijms16011755 - 13 Jan 2015
Cited by 7 | Viewed by 8024
Abstract
Alternative splicing is a pervasive mechanism of RNA maturation in higher eukaryotes, which increases proteomic diversity and biological complexity. It has a key regulatory role in several physiological and pathological states. The diffusion of Next Generation Sequencing, particularly of RNA-Sequencing, has exponentially empowered [...] Read more.
Alternative splicing is a pervasive mechanism of RNA maturation in higher eukaryotes, which increases proteomic diversity and biological complexity. It has a key regulatory role in several physiological and pathological states. The diffusion of Next Generation Sequencing, particularly of RNA-Sequencing, has exponentially empowered the identification of novel transcripts revealing that more than 95% of human genes undergo alternative splicing. The highest rate of alternative splicing occurs in transcription factors encoding genes, mostly in Krüppel-associated box domains of zinc finger proteins. Since these molecules are responsible for gene expression, alternative splicing is a crucial mechanism to “regulate the regulators”. Indeed, different transcription factors isoforms may have different or even opposite functions. In this work, through a targeted re-analysis of our previously published RNA-Sequencing datasets, we identified nine novel transcripts in seven transcription factors genes. In silico analysis, combined with RT-PCR, cloning and Sanger sequencing, allowed us to experimentally validate these new variants. Through computational approaches we also predicted their novel structural and functional properties. Our findings indicate that alternative splicing is a major determinant of transcription factor diversity, confirming that accurate analysis of RNA-Sequencing data can reliably lead to the identification of novel transcripts, with potentially new functions. Full article
(This article belongs to the Special Issue Pre-mRNA Splicing)
Show Figures

Figure 1

Figure 1
<p>Computational and experimental workflow. Schematic overview of the <span class="html-italic">in silico</span> procedures used to infer the presence of new transcription factors (TFs) transcripts from the re-analysis of our RNA-Seq datasets. The experimental approach used to validate the presence of the new variants is also depicted.</p>
Full article ">Figure 2
<p>Schematic representation of newly identified TF transcripts. Newly identified transcripts encoding TFs (black)—<span class="html-italic">ZNF266</span>, <span class="html-italic">SATB1</span>, <span class="html-italic">ELF2</span>, <span class="html-italic">SP140L</span>, <span class="html-italic">ARID5B</span>, <span class="html-italic">NCOA2</span> and <span class="html-italic">IRF1</span>—are schematically compared to known gene annotations: RefSeq (blue), AceView predictions (purple) and Gencode (red).</p>
Full article ">Figure 3
<p>Graphical representation of newly identified splicing events in TF genes. General scheme of the new alternative splicing events identified for <span class="html-italic">SATB1</span> (<b>A</b>); <span class="html-italic">ELF2</span> (<b>B</b>); <span class="html-italic">SP140L</span> (<b>C</b>); <span class="html-italic">ARID5B</span> (<b>D</b>); <span class="html-italic">NCOA2</span> (<b>E</b>) and <span class="html-italic">ZNF266</span> (<b>F</b>). For all the genes, the genomic region encompassing the gene is shown in the upper part. Nucleotide sequences (and electropherograms by Sanger sequencing) of the new splice junctions are shown below the exon/intron structure for each gene. White numbers indicate exons’ numbers. Donor and acceptor splice sites are shown in bold. Red arrows indicate the primers annealing sites.</p>
Full article ">Figure 4
<p>Multiple alignment of the <span class="html-italic">N</span>-terminal residues of the novel predicted ZNF266 variants. The evolutionary conservation of the new 67 amino acids is shown in (<b>A</b>); Krüppel-associated box (KRAB)-A and -B boxes (dashed lines) alignment with other human genes is shown in (<b>B</b>). Identical residues are indicated by “<b>*</b>”, conservation between groups of strongly and weakly similar properties by “:” and “.” respectively. Black arrow indicates the start of the canonical ZNF266 protein isoform.</p>
Full article ">Figure 5
<p><span class="html-italic">In silico</span> characterization of ZNF266 predicted protein. Panel <b>A</b> shows relative surface accessibility (RSA) per residue; 3D structure of both annotated (<b>left</b>) and new (<b>right</b>) ZNF266 isoforms are shown in panel <b>B</b>. The protein backbone is shown in grey. The arrows show the direction of the beta-sheets, which is from the <span class="html-italic">N</span>- to the <span class="html-italic">C</span>-terminus. In both panels, functional domains and motifs are highlighted. In particular, the KRAB domain of the new ZNF266 isoform is shown in green, three predicted additional ZNF motifs in orange and the annotated ZNF motifs in yellow.</p>
Full article ">
Previous Issue
Next Issue
Back to TopTop